MX2013000395A - Methods of generating natural killer cells. - Google Patents

Abstract

Provided herein are methods of producing natural killer cells using a two-step expansion and differentiation method. Also provided herein are methods of suppressing tumor cell proliferation, of treating individuals having cancer or a viral infection, comprising administering the NK cells produced by the method to an individual having the cancer or viral infection.

Description

METHODS OF GENERATING NATURAL KILLING CELLS 1. RELATED REQUESTS This application claims the benefit of the provisional patent application of E.U.A. Serial No. 61 / 363,981, filed July 13, 2010 and provisional patent application of E.U.A. Serial No. 61 / 497,897, filed on June 16, 2011, the descriptions of which are hereby incorporated by reference in their entirety. 2. FIELD OF THE INVENTION Methods of producing a population of natural killer cells, eg, natural killer cells derived from placenta, eg, placental perfusate (eg, perfused human placenta) such as natural killer cells derived from placenta, are provided herein. , or other tissues, for example, umbilical cord blood or peripheral blood. Also present are populations of expanded natural killer cells produced by the methods presented herein. In addition, methods of using placental perfusate, and natural killer cells thereof, to suppress the proliferation of tumor cells are provided herein. In certain embodiments, natural killer cells are used in combination with, or treated with, one or more immunomodulatory compounds, for example, immunomodulatory compounds referred to as IMiDs ™. 3. BACKGROUND OF THE INVENTION Natural killer (NK) cells are cytotoxic lymphocytes that are a major component of the innate immune system. NK cells do not express receptors for T-cell antigen (TCR), CD3 or B-cell surface immunoglobulin receptor (Ig). NK cells usually express the surface markers CD16 (FcyRIII) and CD56 in humans, but a subclass of human NK cells is CD16. NK cells are cytotoxic; Small granules in your cytoplasm contain special proteins such as perforin and proteases known as granzymes. When released in close proximity to a cell targeted for killing, perforin forms pores in the cell membrane of the target cell through which granzymes and associated molecules can enter, inducing apoptosis. A granzyme, granzyme B (also known as granzyme 2 and serine esterase 1 associated with cytotoxic T lymphocyte), is a serine protease crucial for rapid induction of target cell apoptosis in the cell-mediated immune response.

NK cells are activated in response to interferons or cytokines derived from macrophages. Activated NK cells are referred to as killer cells activated with lymphokine (LAK). NK cells possess two types of surface receptors, labeled "activating receptors" and "inhibitory receptors" that control the cytotoxic activity of cells.

Among other activities, NK cells play a role in tumor host rejection. Since cancer cells have reduced expression or none of MHC class I, they become targeted by NK cells. Accumulating clinical data suggest that haploidentic transplantation of human NK cells isolated from peripheral blood mononuclear cells (PBMC) or potent antileukemia effects mediated bone marrow without detectable graft that incurs host disease (GVHD). See uggeri et al., Science 295: 2097-2100 (2002). Natural killer cells can be activated by cells lacking, or exhibiting reduced levels of, major histocompatibility complex (MHC) proteins. Additionally, activating receptors expressed in NK cells are known to mediate the detection of cells "stressed" or transformed with express ligands to activating receptors and thus trigger NK cell activation. For example, NCR1 (NKp46) binds viral hemagglutinins. Ligands of NKG2D include CMV binding protein 1 UL16 (ULB1), ULB2, ULB3 and sequence A related to MHC class I polypeptide (MICA) and MICB proteins. The protein NK 2B4 binds CD48, and DNAM- a poliovirus receptor (PVR) and Nectin-2, both are consistently connected in acute myeloid leukemia (AML). See Penda et al., Blood 105: 2066-2073 (2004). In addition, AML lysis has been described primarily as a natural cytotoxicity (NCR) receptor dependent. See Fauriat et al., Blood 109: 323-330 (2007). Activated and expanded NK cells and peripheral blood LAK cells have been used in ex vivo therapy and in vivo treatment of patients who have advanced cancer, with some success against bone marrow related diseases, such as leukemia.; breast cancer; and certain types of lymphoma. LAK cell treatment requires that the patient first receive IL-2, followed by leukopheresis and then an ex vivo incubation and culture of autologous lymphocytes harvested in the presence of IL-2 for a few days. LAK cells are due to complete therapy. This purging treatment is expensive and can cause serious side effects. These include fluid retention, pulmonary edema, drop in blood pressure, and high fever.

Despite the advantageous properties of NK cells in killer tumor cells and virus infected cells, they remain difficult to work with and apply in immunotherapy, primarily because of the difficulty of maintaining their tumor and tumoricidal targeting abilities during culture and expression. In this way, it is necessary in the art to develop an efficient method to produce and expand natural killer cells that retain tumoricidal functions.

BRIEF DESCRIPTION OF THE INVENTION In the present methods are provided for expanding and differentiating cells, for example, hematopoietic cells, such as hematopoietic stem cells, for example, CD34 + hematopoietic stem cells, to natural killer cells. In one aspect, a method of producing natural killer (NK) cells comprising culturing hematopoietic stem cells or progenitor cells, e.g., stem cells or CD34 + progenitor cells, is provided in a first medium to produce expanded and differentiated cells, and subsequently cultivating said expanded cells in a second medium wherein said cells expand further and differentiate into natural killer cells. The first and second steps involve cultivating the cells in medium with a unique combination of cellular factors. In certain embodiments, said cellular factors (e.g., cytokines) are not comprised within an undefined component of the medium (e.g., serum), e.g., cellular factors (e.g., cytokines) are exogenous to the undefined component of the medium (for example, serum). In certain modalities, said method is a two-step method. In certain embodiments, said method does not comprise any third step or intermediate where the cells are contacted. In a specific embodiment, a method of producing a population of activated natural killer (NK) cells is provided herein, comprising: (a) planting a population of stem cells or hematopoietic progenitors in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of the stem cell factor (SCF) and interleukin-7 (IL-7), wherein said optional IL-15 and SCF and IL-7 are not comprised within an undefined component of said medium, so that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; and (b) expanding the cells of the first passage in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells. Natural killer cells produced by the methods provided herein (eg, two-step method) are referred to herein as TSNK cells.

In certain embodiments, said first medium comprises medium comprising one or more of human serum (e.g., human serum AB), fetal bovine serum (FBS) or fetal calf serum (FCS), e.g., 5% to 20% v / v, stem cell factor (SCF), for example, 1 ng / mL at 50 ng / mL, tyrosine kinase-3 ligand of FMS type (ligand Flt-3), for example, 1 ng / ml at 20 ng / mL; interleukin-7 (IL-7), for example, 1 ng / mL at 50 ng / mL; thrombopoietin (TPO), for example, 1 ng / mL at 50 ng / mL; Interleukin-2 (IL-2), for example, 50 IU / mL at 500 IU / mL; interleukin-15 (IL-5), for example, 1 ng / mL at 50 ng / mL; and / or heparin, for example, 0.1 lU / mL at 10 lU / mL. In a specific embodiment, said first medium comprises the stem cell factor (SCF), interleukin-7 (I L-7) and interleukin-15 (IL-15). In another specific embodiment, said first medium comprises growth medium, human serum (e.g., human serum AB), FBS FCS, SCF, IL-7 and IL-15. In another specific embodiment, said first medium further comprises ligand Flt-3 (Flt3-L), IL-2 and / or heparin. In another specific embodiment, said first medium comprises growth medium, 10% human serum or fetal bovine serum., 20 ng / mL SCF, 10 ng / ml Flt3-L, 20 ng / mL IL-7, 20 ng / mL TPO, 200 lU / mL IL-2, 10 ng / mL IL-15, and 1.5 lU / mL heparin. In another specific embodiment, said first medium does not comprise IL-2. In another specific embodiment, said culture in said first medium comprises culture using feeder cells, e.g., K562 cells, e.g., K562 cells treated with mitomycin C, peripheral blood mononuclear cells (PBMCs), e.g., PBMCs treated with mitomycin C or tissue culture adherent stem cells, e.g., tissue culture adherent stem cells treated with mitomycin C.

In certain embodiments, said first medium is, or comprises, GBGM®, AIM-V®, X-VIVO ™ 10, X-VIVO ™ 15, OPTIMIZER, STEMSPAN® H3000, CELLGRO COMPLETE ™, and / or DMEM.F12. In certain embodiments, said medium comprises O-acetyl-carnitine (also referred to as acetylcarnitine, O-acetyl-L-carnitine or OAC), for example, about 0.5 mM-10 mM. In one embodiment, said medium comprises Stemspan® H3000 and / or DMEM: F12 and OAC, for example, about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM. In a specific embodiment, said means comprises GBGM®. In another specific embodiment, said medium comprises DMEM.F12 and approximately 5 mM OAC. In another specific embodiment, said medium comprises Stemspan® H3000 and approximately 5 mM OAC.

In certain embodiments, said second medium comprises cell growth medium comprising one or more of: human serum (eg, human serum AB), fetal bovine serum (FBS) or fetal calf serum (FCS), for example, 5% - 15% FCS v / v; IL-2, for example, 10 IU / mL at 1000 lU / mL; transferin, for example, 10 μ? /? t ?? at 50 μg mL; insulin, for example, 5 μg / mL at 20 μg mL; ethanolamine, for example, 5 x 10"4 to 5 x 10" 5 M; oleic acid, for example, 0.1 μg / mL at 5 μg / mL; linoleic acid, for example, 0.1 μg / mL at 5 μg / mL; palmitic acid, for example, 0.005 μg / ml_ at 2 μg / mL; bovine serum albumin (BSA), for example, 1 μ? / ???. at 5 μg / mL; and / or phytohemagglutinin, for example, 0.01 μg / mL to 1 μg / mL. In a specific embodiment, said second medium comprises IL-2. In a more specific embodiment, said second medium comprises cell growth medium comprising human serum, FBS or FCS, for example, 10% v / v, IL-2, transferin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid. , bovine serum albumin (BSA) and / or phytohemagglutinin. In a more specific embodiment, said second medium comprises Iscove's modified Dulbecco's medium (IMDM), 10% human serum, FBS or FCS, 400 IU IL-2, 35 μg / mL insulin, 2 x 10"5 M ethanolamine, 1 μg / mL oleic acid, 1 μg / mL linoleic acid, 0.2 μ? / Gp? palmitic acid, 2.5 μg / mL BSA and 0.1 ng / mL phytohaemagglutinin In another specific modality, said culture in said second medium comprises cultivating using feeder cells, for example, K562 cells (e.g., K562 cells treated with mitomycin C) or PBMCs (e.g., PBMC treated with mitomycin C), e.g., at the time cells are initiated in said second medium, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days later In certain embodiments, said medium comprises GBGM®, AIM-V®, X-VIVO ™ 10, X-VIVO ™ 15, OPTMIZER, STEMSPAN® H3000, CELLGRO COMPLETE ™, and / or DMEM: F12 In certain embodiments, said medium comprises one or more of O-acetyl-carnitine (also referred to as acetylcanitine, O-acetyl-L-carnit ina or OAC), or a compound that affects acetyl-CoA cyclization in mitodronia, thiazo- vivine, Y-27632, "py i ntegri n", Rho kinase inhibitors (ROCK), caspase inhibitors, or other anti-retroviral compounds / peptides. apoptotic, NOVA-RS (Sheffield Bio-Science) or other small molecule growth enhancers. In certain embodiments, said medium comprises nicotinamide. In certain embodiments, said medium comprises about 0.5 mM-10 mM OAC. In one embodiment, said medium comprises Stemspan® H3000, and / or DMEM: F12 and OAC, for example, about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM. In a specific embodiment, said medium comprises GBGM®. In another specific embodiment, said medium comprises DMEM: F12 and approximately 5 mM OAC. In another specific embodiment, said medium comprises Stemspan® H3000 and approximately 5 mM OAC.

In another specific embodiment, a method of producing a population of activated natural killer (NK) cells is provided, comprising: (a) planting a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 ( IL-15) and, optionally, one or more stem cell (SCF) and interleukin-7 (IL-7) factors, wherein said IL-15 and optionally SCF and IL-7 are not comprised within an undefined component of said medium, so that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.

In another specific embodiment, a two-step method of producing a population of natural killer (NK) cells is provided herein, wherein a first step of said method comprises expanding a population of hematopoietic stem or progenitor cells into a first medium that comprises one or more of SCF, IL-7 and IL-15, and wherein said SCF, IL-7 and IL-15 are not comprised within an undefined component of said medium (e.g., serum), and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; and wherein a second step of said method comprises expanding the cells from the first passage in a second medium comprising IL-2, to produce activated NK cells. In another specific embodiment, said first medium further comprises one or more of tyrosine kinase 3 ligand of the Fms type (FU3-L), thrombopoietin (Tpo), interleukin-2 (IL-2), and / or heparin. In another specific embodiment, said first medium further comprises about 5% -20% of fetal bovine serum or human serum. In another specific embodiment, the SCF is present at a concentration of about 1 to about 50 ng / mL in the first medium. In another specific embodiment, F 113-L is present at a concentration of about 1 to about 150 ng / mL in the first medium. In another specific embodiment, IL-2 is present at a concentration of about 50 to about 1500 IU / mL in the first medium. In another specific embodiment, IL-7 is present at a concentration of from about 1 to about 150 ng / mL in the first medium. In another specific embodiment, IL-15 is present at a concentration of from about 1 to about 150 ng / mL in the first medium. In another specific embodiment, the TPO is present at a concentration of about 1 to about 150 ng / mL in the first medium. In another specific embodiment, heparin is present at a concentration of about 0.1 to about 30 U / mL in the first medium. In another specific embodiment, the IL-2 in the second step is present at a concentration of about 50 to about 1500 IU / mL in the second medium. In another specific embodiment, said second means additionally comprises one or more of fetal calf serum (FCS), transferin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin (BSA) and phytohemagglutinin.

In certain specific embodiments, said hematopoietic stem or progenitor cells are cultured in said first medium during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days before said cultivation in said second medium. In certain other specific embodiments, said cells are cultured in said second medium during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. In a more specific embodiment, said hematopoietic stem or progenitor cells are cultured in said first medium for 21 days, and then cultured in said second medium for 21 days.

In addition, a population of natural killer cells produced by the two-step method described above, referred to herein as TSNK cells, is provided herein. In a specific embodiment, said NK cells (e.g., TSNK cells) are CD3"CD56 +." In a specific embodiment, said NK cells (e.g., TSNK cells) are CD3"CD56 + CD 16." In another specific embodiment, said NK cells (e.g., TSNK cells) are additionally CD94 + CD117 + In another specific embodiment, said NK cells (e.g., TSNK cells) are additionally CD161". In another specific embodiment, said NK cells (e.g., TSNK cells) are additionally NKG2D +. In another specific embodiment, said NK cells are additionally NKp464. In another specific embodiment, said NK cells are additionally CD226 +.

In certain modalities, more than 90%, 92%, 94%, 96% or 98% of said TSNK cells are CD56 + and CD16. "In some modalities, at least 80%, 82%, 84%, 86%, 88 % or 90% of said TSNK cells are CD3"and CD56 +. In other modalities, more than 90%, 92%, 94%, 96% or 98% of said TSNK cells are CD56 +, CD16 and CD3 \ In other modalities, at least 50%, 52%, 54%, 56% , 58% or 60% of said TSNK cells are NKG2D +. In other embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of said TSNK cells are NKB1 +. In certain other embodiments, less than 10%, 8%, 6%, 4% or 2% of said TSNK cells are CD56 + and CD16 +. In more specific modalities, at least 50%, 55%, 60%, 65% or 70% of said TSNK CD3"cells, CD56 + are NKp46 +, in other more specific modalities, at least 50%, 55%, 60 %, 65%, 70%, 75%, 80% or 85% of said TSNK CD3"cells, CD56 + are CD117 +. In other more specific embodiments, at least 20%, 25%, 30%, 35%, 40% or 45% of said TSNK CD3"cells, CD56 + are CD94 +, in other more specific modalities, at least 10%, 20 %, 25%, 30%, 35%, 40%, 45% or 50% of said TSNK CD3"cells, CD56 + are CD161." In other more specific modalities, at least 10%, 12%, 14%, 16 %, 18% or 20% of said TSNK CD3", CD56 * cells are CD226 +. In more specific embodiments, at least 20%, 25%, 30%, 35% or 40% of said TSNK CD3", CD56 + cells are CD7 +, in more specific embodiments, at least 30%, 35%, 40 %, 45%, 50%, 55% or 60% of said TSNK CD3", CD56 + are CD5 +.

In another aspect, the use of TSNK cells is provided herein to suppress tumor cell proliferation, treat viral infection or treat cancer, for example, blood cancers and solid tumors. In certain embodiments, the TSNK cells make contact with, or are used in combination with, an immunomodulatory compound, for example, an immunomodulatory compound described in section 6.2.1 below, or thalidomide.

In a specific embodiment, said cancer is a solid tumor. In another embodiment, said cancer is a blood cancer. In specific modalities, the cancer is glioblastoma, primary ductal carcinoma, leukemia, acute T cell leukemia, chronic myeloid lymphoma (CML), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), lung carcinoma, colon adenocarcinoma, histiocytic lymphoma, colorectal carcinoma, colorectal adenocarcinoma, prostate cancer, multiple myeloma or retinoblastoma.

In another specific embodiment, hematopoietic cells, for example hematopoietic progenitor or stem cells, of which TSNK cells are produced, are obtained from perfusate of placenta, umbilical cord blood or peripheral blood. In another specific embodiment, hematopoietic cells, e.g., hematopoietic progenitor or stem cells, of which TSNK cells are produced, are combined placental perfusate and cord blood cells, e.g., cord blood from the same placenta as the perfused one In another specific embodiment, said umbilical cord blood is isolated from a placenta different from the placenta from which said placental perfusate is obtained. In certain embodiments, the combined cells can be obtained by pooling or combining cord blood and placental perfusate. In certain modalities, cord blood and placental perfusate are combined at a ratio of 100: 1, 95: 5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 1: 1, 1: 5, 1:10, 1:15, 1:20, 1: 25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1: 100, or similar by volume to obtain the combined cells. In a specific embodiment, cord blood and placental perfusate are combined at a ratio of 10: 1 to 1:10, 5: 1 to 1: 5, or 3: 1 to 1: 3. In other specific embodiments, cord blood and placental perfusate are combined at a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10. In a more specific modality, cord blood and placental perfusate are combined at a ratio of 8.5: 1.5 (85%: 15%).

In certain modalities, cord blood and placental perfusate are combined at a ratio of 100: 1, 95: 5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 1: 1, 1: 5, 1:10, 1:15, 1:20. 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1: 85, 1:90, 1:95, 1: 100, or similar by total nucleated cell (TNC) content to obtain the combined cells. In a specific embodiment, cord blood and placental perfusate are combined at a ratio of 10: 1 to 10: 1, 5: 1 to 1: 5, or 3: 1 to 1: 3. In another specific embodiment, cord blood and placental perfusate are combined at a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10.

In one embodiment, therefore, a method of treating an individual having cancer or a viral infection, which comprises administering to said individual an effective amount of isolated TSNK cells, is provided herein.

In a specific embodiment, the isolated TSNK cells have been treated with an immunomodulatory compound, for example, an immunomodulatory compound described in section 6.2.1 below, or thalidomide, prior to said administration. In another specific embodiment, the method comprises administering to the individual (1) an effective amount of isolated TSNK cells; and (2) an effective amount of an immunomodulatory compound or thalidomide. An "effective amount" in this context means an amount of TSNK cells, and optionally immunomodulatory compound or thalidomide, which results in a detectable improvement in one or more symptoms of said cancer or said infection, in comparison with an individual having said cancer or said infection that has not been administered said TSNK cells and, optionally, an immunomodulatory compound or thalidomide. In a specific embodiment, said immunomodulatory compound is lenalidomide or pomalidomide. In another embodiment, the method further comprises administering an anti-cancer compound to the individual, for example, one or more of the anti-cancer compounds described in section 6.8.3, below.

In another embodiment, a method of suppressing the proliferation of tumor cells comprising contacting the tumor cells with a therapeutically effective amount of TSNK cells is provided herein.

In a specific embodiment, the isolated TSNK cells have been treated with an immunomodulatory compound, for example, an immunomodulatory compound described in section 6.2.1 below, or thalidomide, prior to said contact. In another specific embodiment, the tumor cells are additionally contacted with an effective amount of an immunomodulatory compound, for example, an immunomodulatory compound described in section 6.2.1 below, or thalidomide. An "effective amount" in this context means an amount of TSNK cells, and optionally an immunomodulatory compound or thalidomide, which results in a detectable suppression of said tumor cells compared to an equivalent number of tumor cells not contacted with said TSNK cells, and optionally an immunomodulatory compound or Thalidomide In another specific embodiment, the method further comprises contacting the tumor cells with an effective amount of an anti-cancer compound, for example, an anti-cancer compound described in section 6.8.3 below.

In a specific embodiment of this method, the tumor cells are cancer lymphocytes. In another specific embodiment, the tumor cells are solid tumor cells. In another embodiment, the tumor cells are primary ductal carcinoma cells, leukemia cells, acute T cell leukemia cells, chronic myeloid lymphoma (CML) cells, acute myelogenous leukemia cells, chronic myelogenous leukemia (CML) cells, glioblastoma cells, lung carcinoma cells, colon adenocarcinoma cells, histiocytic lymphoma cells, multiple myeloma cells, retinoblastoma cells, colorectal carcinoma cells, prostate cancer cells, or colorectal adenocarcinoma cells. In another specific modality, said contact occurs in vitro. In another specific modality, said contact occurs in vivo. In a more specific modality, said in vivo contact occurs in a human.

In another aspect, herein is provided a method of treating an individual having multiple myeloma, comprising administering to the individual (1) lenalidomide; (2) melphalan; and (3) expanded NK cells, wherein said NK cells are effective to treat multiple myeloma in said individual. In a specific embodiment, said NK cells are NK cells of cord blood, or NK cells produced from cord blood hematopoietic cells, e.g., hematopoietic stem cells. In another embodiment, said NK cells have been produced by any of the methods described herein to produce NK cells, for example, to produce TSNK cells. In another embodiment, said NK cells have been expanded prior to said administration. In another embodiment, said lenalidomide, melphalan, and / or NK cells are administered separately from each other. In certain specific embodiments of the method of treating an individual with multiple myeloma, said NK cells are produced by a two-step method of producing a population of activated natural killer (NK) cells, wherein a first step of said method comprises expanding a population. of hematopoietic stem or progenitor cells in a first medium comprising one or more of the stem cell factor (SCF), interleukin-7 (I L-7) and interleukin-15 (IL-15), and wherein said SCF, IL-7 and IL-15 are not comprised within an undefined component of said medium, and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; and wherein a second step of said method comprises expanding the cells from the first step into a second medium comprising interleukin-2 (IL-2), to produce activated NK cells.

In other specific modalities of the method of treating an individual with multiple myeloma, said NK cells are produced by a method comprising: (a) sowing a population of stem cells or hematopoietic progenitors in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of the stem cell factor (SCF) and interleukin-1 7 (IL-7), wherein said optional IL-15 and SCF and IL-7 are not comprised within an undefined component of said medium, such that the population expands, and a plurality of said population of stem cells or Hematopoietic progenitors differentiate into NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.

In another aspect, a method of treating an individual having chronic lymphocytic leukemia (CLL), which comprises administering to the individual a therapeutically effective dose of (1) lenalidomide, is provided; (2) melphalan; (3) Fludarabine; and (4) expanded NK cells, for example, TSNK cells, wherein said NK cells are effective to treat said CLL in said individual. In a specific embodiment, said NK cells are NK cells of cord blood, or NK cells produced from cord blood hematopoietic cells, e.g., hematopoietic stem cells. In another embodiment, said NK cells have been produced by any of the methods described herein to produce NK cells, for example, to produce TSNK cells. In a specific embodiment of any of the above methods, said NK cells have expanded for at least 10 days before said administration. In a specific embodiment of any of the above methods, said lenalidomide, melphalan, fludarabine and expanded NK cells are administered to said individual separately. In certain specific embodiments of the method of treating an individual with CLL, said NK cells are produced by a two-step method of producing a population of activated natural killer (NK) cells, wherein a first step of said method comprises expanding a population of hematopoietic stem or progenitor cells in a first medium comprising one or more of the stem cell factor (SCF), interleukin-7 (IL-7) and interleukin-15 (IL-15), and wherein said SCF, IL-7 and IL-15 are not comprised within an undefined component of said medium, and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; and wherein a second step of said method comprises expanding the cells the first step into a second medium comprising interleukin-2 (IL-2), to produce activated NK cells.

In other specific embodiments of the method of treating an individual with CLL, said NK cells are produced by a method comprising: (a) planting a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of stem cell factor (SCF) and interleukin-7 (IL-7), wherein said optional IL-15 and SCF and IL-7 are not comprised within an undefined component of said medium , so that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.

In one aspect, a method of cryopreservating a population of NK cells, e.g., TSNK cells, is provided herein. In one embodiment, said method comprises: (a) sowing a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-IL (IL-15) and, optionally, one or more of the stem cell factor (SCF) and interleukin-7 (IL-7), wherein said optional IL-15 and SCF and IL-7 are not comprised within an undefined component of said medium, so that the population expands, and a plurality of stem cells or Hematopoietic progenitors within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells, and (c) cryopreservating the NK cells of step (b) in a means of cryopreservation. In a specific embodiment, said step (c) further comprises (1) preparing a cell suspension solution; (2) adding cryopreservation medium to the cell suspension solution of step (1) to obtain cryopreserved cell suspension; (3) cooling the cryopreserved cell suspension of step (3) to obtain a cryopreserved sample; and (4) classify the cryopreserved sample below -80 ° C. In certain embodiments, the method includes any intermediate step between step (a) and (b), and between step (b) and (c).

In another embodiment, said method of cryopreservating a population of NK cells, for example, TSNK cells comprises: (a) expanding a population of hematopoietic stem or progenitor cells into a first medium comprising one or more of the stem cell factor (SCF) , IL-2, interleukin-7 (IL-7), interleukin-15 (IL-15) and heparin, and wherein said SCF, IL-2, IL-7 and IL-15 are not comprised within a non-component. defined from said medium, and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells; and (c) cryopreservating the NK cells of step (b) in a cryopreservation medium. In a specific embodiment, said step (c) further comprises (1) preparing a cell suspension solution; (2) adding cryopreservation medium to the cell suspension solution of step (1) to obtain cryopreserved cell suspension; (3) cooling the cryopreserved cell suspension of step (3) to obtain a cryopreserved sample; and (4) store the cryopreserved sample below -80 ° C. In certain embodiments, the method includes any intermediate step between step (a) and (b), and between step (b) and (c), and / or no additional culture step before step (a).

In another specific embodiment, hematopoietic cells, e.g., hematopoietic progenitor or stem cells of which TSNK cells are produced express one or more of microRNAs hsa-mR-380, hsa-miR-512, hsa-miR-517 , hsa-miR-518c, hsa-miR-519b, and hsa-miR-520a at a detectably higher level than the natural killer cells of peripheral blood, as determined, for example, by quantitative real-time PCR (qRT- PCR).

In another specific embodiment of the above methods, said TSNK cells are contacted with an immunomodulatory compound or thalidomide in an amount and for a time sufficient for said natural killer cells to detectably express more granzyme B or perforin than an equivalent number of natural killer cells, for example, TSNK cells, not contacted with said immunomodulatory compound or thalidomide. In another specific embodiment, said TSNK cells are contacted with an immunomodulatory compound or thalidomide in an amount and for a time sufficient for said cells to detectably express more cytotoxicity to said tumor cells than an equivalent amount of natural killer cells, eg, TSNK cells. , not contacted with said immunomodulatory compound, for example, lenalidomide or pomalidomide, or with thalidomide. In other specific modality, said TSN cells express one or more of BAX, CCL5, CCR5, CSF2, FAS, GUSB, IL2RA or TNFRSF18 at a level higher than an equivalent number of natural killer cells, eg, TSNK cells, not contacted with said immunomodulatory compound or thalidomide. In another specific embodiment, said TSNK cells express one or more of ACTB, BAX, CCL2, CCL3, CCL5, CCR5, CSF1, CSF2, ECE1, FAS, GNLY, GUSB, GZMB, IL1A, IL2RA, IL8, IL10, LTA. , PRF1, PTGS2, SK1 and / or TBX21 at a level higher than an equivalent number of natural killer cells, e.g., TSNK cells, not contacted with said immunomodulatory compound or thalidomide.

In certain embodiments of the above tumor treatment or suppression methods, the TSNK cells are combined with other natural killer cells, eg, natural killer cells isolated from placental perfusate, umbilical cord blood or peripheral blood, or produced from hematopoietic cells by a different method In specific embodiments, the TSNK cells are combined with natural killer cells from another source, or made by a different method, in a ratio of approximately 100: 1, 95: 5, 90:10, 85:15, 80:20, 75 : 25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85 , 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50 : 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 1: 1, 1: 5, 1:10 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1 : 75, 1:80, 1:85, 1:90, 1:95, 1: 100, or similar.

In another aspect, a comparison comprising isolating TSNK cells is provided herein. In a specific embodiment, said TSNKs are produced from hematopoietic cells, for example, hematopoietic progenitor or stem cells isolated from placental perfusate, umbilical cord blood, and / or peripheral blood. In another specific embodiment, said TSNK cells comprise at least 50% cells in the composition. In another specific embodiment, said TSNK cells comprise at least 80%, 85%, 90%, 95%, 98% or 99% of cells in the composition. In certain modalities, more than 90%, 92%, 94%, 96% or 98% of TSNK cells in said composition are CD56 + and CD16. "In other modalities, at least 80%, 82%, 84%, 86% , 88% or 90% of TSNK cells in said composition are CD3"and CD56 +. In other embodiments, at least 50%, 52%, 54%, 56%, 58% or 60% of said cells are NKG2D *. In other embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of said cells are NKB 1 +. In certain other embodiments, less than 10%, 8%, 6%, 4% or 2% of said TSNK cells are NKAT2 +. In certain other embodiments, less than 10%, 8%, 6%, 4% or 2% of said TSNK cells are CD56 + and CD16 +. In more specific modalities, at least 50%, 55%, 60%, 65% or 70% of said TSNK CD3"cells, CD56 + are NKp46 +, in other more specific modalities, at least 50%, 55%, 60 %, 65%, 70%, 75%, 80% or 85% of said TSNK CD3"cells, CD56 + are CD117 +. In other more specific modalities, at least 20%, 25%, 30%, 35%, 40% or 45% of said TSNK CD3"cells, CD56 + are CD94 +, in other more specific modalities, at least 10%, 12 %, 14%, 16%, 18% or 20% of said TSNK CD3, CD56 + cells are CD226 \ In more specific modalities, at least 20%, 25%, 30%, 35% or 40% of said TSNK CD3 cells ", CD56 + are CD7." In more specific modalities, at least 30%, 35%, 40%, 45%, 50%, 55% or 60% of said TSNK CD3, CD56 + cells are CD5 +.

In another specific embodiment, said TSNK CD56 +, CD16"cells are from a single individual, In a more specific embodiment, said natural killer cells CD56 +, CD16" comprise natural killer cells from at least two different individuals. In another specific embodiment, said TSNK cells have been contacted with an immunomodulatory compound or thalidomide in an amount and for a time sufficient for said TSNK cells to detectably express more granzyme B or perforin than an equivalent number of natural killer cells, i.e. TSNK, not contacted with said immunomodulatory compound or thalidomide. In another specific embodiment, said composition additionally comprises an immunomodulatory compound or thalidomide. In certain embodiments, the immunomodulatory compound is a compound described in section 6.2.1 below, for example, an amino-substituted isoindoline compound.

In another specific embodiment, the composition additionally comprises one or more anti-cancer compounds, for example, one or more of the anti-cancer compounds described in section 6.8.2 below.

In a more specific embodiment, the composition comprises TSNK cells and natural killer cells from another source, or made by another method. In a specific embodiment, said other source is placental blood and / or umbilical cord blood. In another specific embodiment, said other source is peripheral blood. In more specific modalities, TSNK cells are combined with natural killer cells from another source, or made by another method in a ratio of approximately 100: 1, 95: 5, 90:10, 85:15, 80:20, 75: 25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 1: 1, 1: 5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1: 75, 1:80, 1:85, 1:90, 1:95, 1: 100, or similar.

In another specific embodiment, the composition comprises TSNK cells and isolated placental perfusate or isolated placental perfusate cells. In a more specific embodiment, said placental perfusate is from the same individual as said TSNK cells. In another more specific embodiment, said placental perfusate comprises placental perfusate from an individual different from said TSNK cells. In another specific embodiment, all, or substantially all (eg, more than 90%, 95%, 98% or 99%) of the cells in said placental perfusate are fetal cells. In another specific embodiment, placental perfusate or placental perfusate cells comprise fetal and maternal cells. In a more specific embodiment, the fetal cells in said placental perfusate comprise less than about 90%, 80%, 70%, 60% or 50% of the cells in said perfusate. In another specific embodiment, said perfusate is obtained by passing a 0.9% NaCl solution through the placental vasculature. In another specific embodiment, said perfusate comprises a culture medium. In another specific embodiment, said perfusate has been treated to eliminate erythrocytes. In another specific embodiment, said composition comprises an immunomodulatory compound, for example, an immunomodulatory compound described in section 5.2.1.1 below, for example, an amino-substituted isoindoline compound. In another specific embodiment, the composition additionally comprises one or more anti-cancer compounds, for example, one or more of the anti-cancer compounds described in section 6.8.2 below.

In another specific embodiment, the composition comprises TSNK cells and placental perfusate cells. In a more specific embodiment, said placental perfusate cells are from the same individual as said TSNK cells. In another more specific embodiment, said placental perfusate cells are from a different individual than said TSNK cells. In another specific embodiment, the composition comprises isolated placental perfusate and isolated placental perfusate cells, wherein said placental perfusate and said isolated placental perfusate cells are of different individuals. In another modality more specific to any of the above embodiments comprising placental perfusate, said placental perfusate comprises placental perfusate of at least two individuals. In another more specific embodiment of any of the above embodiments comprising placental perfusate cells, said isolated placental perfusate cells are of at least two individuals. In another specific embodiment, said composition comprises an immunomodulatory compound. In another specific embodiment, the composition additionally comprises one or more anti-cancer compounds, for example, one or more of the anti-cancer compounds described in section 6.8.2, below. 4. 1 Definitions As used herein, the terms "immunomodulatory compound" and "I i D ™" do not encompass thalidomide.

As used herein, "lenalidomide" means 3- (4'-aminoisoindoline-1 -one) -1-piperidine-2,6-dione (name of Chemical Abstracts Service) or 2,6-piperidinedione, 3- (4-am ino- 1, 3-dihydro-1-oxo-2H-isoindol-2-yl) - (name of the International Union of Pure and Applied Chemistry (IUPAC)). As used herein, "pomalidomide" means 4-amino-2- (2,6-dioxopiperidin-3-yl) isoindol-1,3-dione.

As used herein, "multipotent", when referring to a cell, means that the cell has the ability to differentiate in a cell from another cell type. In certain embodiments, "a multipotent cell" is a cell that has the ability to grow in any subset of the mammalian body approximately 260 cell types. Unlike a pluripotent cell, a multipotent cell does not have the capacity to form all cell types.

As used herein, "feeder cells" refers to cells of a type that are co-cultured with cells of a second type, to provide an environment in which the cells of the second type can be maintained, and perhaps proliferate. Without being bound by any theory, the feeder cells may provide, for example, peptides, polypeptides, electrical signals, organic molecules (eg, steroids), nucleic acid molecules, growth factors (eg, bFGF), other factors (eg. example, cytokines), and metabolic nutrients to target cells. In certain embodiments, the feeder cells grow in a monolayer.

As used herein, "natural killer cell" or "NK cells" without further modification, includes natural killer cells from any tissue source.

As used herein, "TSNK" and "TSNK cells" refer to natural killer cells produced by the culture / expansion methods (eg, two-step method) described herein.

As used herein, "placental perfusate" means perfusion solution that has been passed through at least part of a placenta, for example, a human placenta, for example, through the placental vasculature, including a plurality of cells harvested by the perfusion solution during the passage through the placenta.

As used herein, "placental perfusate cells" means nucleated cells, e.g., total nucleated cells, isolated from, or that can be isolated from, placental perfusate.

As used herein, "tumor cell suppression", "suppression of tumor cell proliferation", and the like, includes reducing the growth of a population of tumor cells, for example, by killing one or more of the tumor cells in said population of tumor cells, for example, by contacting the population of tumor cells with, for example, TSNK cells or a population of cells comprising TSNK cells.

As used herein, the term "hematopoietic cells" includes hematopoietic stem cells and hematopoietic progenitor cells.

As used herein, the "undefined component" is a term of the art in the field of culture medium which refers to components whose constituents are generally not provided or quantified. Examples of an "undefined component" include, without limitation, human serum (e.g., AB human serum) and fetal serum (e.g., fetal bovine serum or fetal calf serum).

As used herein, "+", when used to indicate the presence of a particular cellular marker, means that the cellular marker is detectably present in fluorescent activated cell sorting over an isotype control; or it is background detectable in quantitative or semi-quantitative RT-PCR.

As used herein, when used to indicate the presence of a particular cellular marker, it means that the cellular marker is not detectably present in fluorescent activated cell sorting over an isotype control; or it is not detectable in the background in quantitative or semi-quantitative RT-PCR. 5. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Fold expansion of differentiated NK cells from hematopoietic stem cells (HSCs) with various medium formulations. The error bars represent standard derivation of three donors. X axis: day (D) of culture. Y axis: fold expansion compared with day 0 (start of culture).

Figure 2: Phenotypic characterization of NK cells cultured with NK2A medium. Cells were labeled in triple with PE-antiCD56, FITC anti-CD3, PerCP-antiCD16. Horizontal, vertical lines: Fluorescent running level significantly background.

Figure 3: Fold expansion of NK cells cultured with NK2A (FF), NK2A (placental stem cells as feeder cells), NK2A (MSC as feeder cells) or two-stage NK medium. X axis: day (D) of culture. Y axis: fold expansion compared with day 0 (start of culture).

Figure 4: Cytotoxicity of NK cells cultured with NK2A (without feeder cells). Two-stage NK medium; NK2A with placental stem cells (PSC) adherent to tissue culture plastic CD34", CD10 \ CD105 \ CD200 + as feeder cells, NK2A with mesenchymal stem cells (MSC) as feeder cells, at post-culture initiation at day 45. The representative data of three donors are shown in Figure 4.

Figure 5: Phenotypic characterization of NK cells on day 41 (D41) of culture. Representative data of 3 individual donors are shown. X axis: percentage of NK cells, produced by the two-step method, which are CD3"CD56 +, CD16" CD56 + or CD16"CD56 +, or that express NKB1, NKG2D, NKp46, CD94, CD117, CD226, CD7 or CD5 Cells were cultured in NK2A (without feeder cells), two-stage NK medium, NK2A with placental stem cells (PSC) adherent to tissue culture plastic CD34", CD10 +, CD105 +, CD200 + as feeder cells, NK2A with mesenchymal stem cells (MSCs) derived from bone marrow as feeder cells, in the post-culture initiation on day 41.

Figure 6: Expression of CD94 and CD117 in the NK cell population CD56 + CD3- during NK culture in NK2A medium. The dominant population of CD56 + CD94 + CD 117 + cells was identified from NK cells cultured in NK2A medium, which can be distinguished from NK cells derived from embryonic stem cell (ESC) (CD56 + CD94 + CD117bai0"). of three donors are shown in Figure 6. X-axis: fluorescence of anti-CD94 labeled with phycoerythrin (PE) Y-axis: anti-CD117 fluorescence labeled with APC Horizontal, vertical lines: fluorescent label level significantly background D13 D20, D28, D35: initiation of cell culture post CD34 + days 13, 20, 28 and 35.

Figure 7: Effects of placental stem cells on cultured NK cells compared to MSC and NK2A medium alone. X axis: NK cells cultured in NK2A medium without a feeder layer (FF); NK cells cultured in NK2A medium with mesenchymal stem cells (MCS) derived from bone marrow as a feeder layer; or NK2A medium with placental stem cells (PDACs) adherent to tissue culture plastic CD10 +, CD34", CD105 +, CD200 + as a feeder layer Y-axis (left): cytotoxicity, expressed as a percentage of remaining tumor cells (1.0 = 100%), cytotoxicity indicated by open boxes, Y axis (right): expansion of NK cell fold using the two-step method, fold expansion expressed as asterisks.

Figures 8A-8B: Effects of relative relationships of umbilical cord blood (UCB) and human placental perfusate (HPP) on the purity of CD34 + post-thaw cells. Figure 8A: Effects of volumetric content of HPP (vol%) in purity of CD34 + Lin. "X axis: volumetric fraction of HPP in the UCB and grouped HPP (combination) Y axis: the percentage of CD34 + Lin cells". Figure 8B: Effects of HPP TNC content (TNC%) on CD34 + Lin purity "Y axis: the percentage of CD34 + Lin cells". 6. DETAILED DESCRIPTION Herein is provided a novel method of producing and expanding NK cells of hematopoietic cells, for example stem cells or hematopoietic progenitor cells. The hematopoietic cells used to produce the NK cells can be isolated from any source, for example, without limitation, placenta, umbilical cord blood, placental blood, peripheral blood, spleen or liver. In a certain embodiment, NK cells are produced from expanded hematopoietic cells, e.g., hematopoietic stem cells and / or hematopoietic progenitor cells. In one embodiment, hematopoietic cells are harvested from a source of said cells, for example, perfused from placenta, umbilical cord blood, peripheral blood, spleen, liver and / or bone marrow. In a specific embodiment, the hematopoietic cells are expanded and differentiated, continuously, in a first medium without the use of feeder cells. The cells are then cultured in a second medium in the presence of feeder cells. Said isolation, expansion and differentiation can be carried out in a central facility, which provides expanded hematopoietic cells for delivery for decentralized expansion and differentiation in points of use, for example, hospital, military base, military front, or similar. 6. 1. Hematopoietic cells The hematopoietic cells useful in the methods described herein can be any hematopoietic cell capable of differentiating into NK cells, for example, precursor cells, hematopoietic progenitor cells, hematopoietic stem cells, or the like. Hematopoietic cells can be obtained from tissue sources such as, for example, bone marrow, cord blood, placental blood, peripheral blood, liver or the like, or combinations thereof. Placental hematopoietic cells can be obtained. In a specific embodiment, the hematopoietic cells are obtained from placental perfusate. The hematopoietic cells of placental perfusate may comprise a mixture of fetal and maternal hematopoietic cells, for example, a mixture wherein maternal cells comprise more than 5% of the total number of hematopoietic cells. Preferably, the perfusate hematopoietic cells of placenta comprise at least about 90%, 95%, 98%, 99% or 99.5% fetal cells.

In another specific embodiment, hematopoietic cells, e.g. stem cells or hematopoietic progenitor cells, from which TSNK cells are produced, are obtained from perfusate of placenta, umbilical cord blood or peripheral blood. In another specific embodiment, hematopoietic cells, e.g., stem cells or hematopoietic progenitor cells, of which TSNK cells are produced, are combined cells from placental perfusate and cord blood, e.g., cord blood from the same placenta as the perfused one In another specific embodiment, said umbilical cord blood is isolated from a placenta different from the placenta from which said placental perfusate is obtained. In certain embodiments, the combined cells can be obtained by pooling or combining cord blood and placental perfusate. In certain modalities, cord blood and placental perfusate are combined at a ratio of 100: 1, 95: 5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 1: 1, 1: 5, 1:10, 1:15, 1:20, 1: 25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1: 100, or similar by volume to obtain the combined cells. In a specific embodiment, cord blood and placental perfusate are combined at a ratio of 10: 1 to 1:10, 5: 1 to 1: 5, or 3: 1 to 1: 3.

In another specific embodiment, cord blood and placental perfusate are combined at a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10. In a more specific modality, cord blood and placental perfusate are combined at a ratio of 8.5: 1.5 (85%: 15%).

In certain modalities, cord blood and placental perfusate are combined at a ratio of 100: 1, 95: 5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 1: 1, 1: 5, 1:10, 1:15, 1:20, 1: 25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1: 100, or similar by total nucleated cell (TNC) content to obtain the combined cells. In a specific embodiment, cord blood and placental perfusate are combined at a ratio of 10: 1 to 10: 1, 5: 1 to 1: 5, or 3: 1 to 1: 3. In another specific embodiment, cord blood and placental perfusate are combined at a ratio of 10: 1, 5: 1, 3: 1, 1: 1, 1: 3, 1: 5 or 1:10.

In another specific embodiment, the hematopoietic cells, for example, stem cells or hematopoietic progenitor cells from which said TSNK cells are produced are from umbilical cord blood and perfused from placenta, but wherein said umbilical cord blood is isolated from a placenta different from the placenta from which said placental perfusate is obtained.

In certain embodiments, the hematopoietic cells are CD34 + cells. In specific embodiments, the hematopoietic cells useful in the methods described herein are CD34 + CD38 + or CD34 + CD38. "In a more specific embodiment, the hematopoietic cells are CD34 + CD38 ~ Lin ~ In another specific embodiment, the hematopoietic cells are one or more of CD2", CD3, CD11b ~, CD11c", CD14", CD16", CD19", CD24", CD56-, CD66b "and / or glycophorin A." In another specific embodiment, the hematopoietic cells are CD2 ', CD3", CD11b", CD11c ", CD14", CD16", CD19", CD24", CD56", CD66b "and glycophorin A." In another more specific modality, the hematopoietic cells are CD34 + CD38"CD33" CD117. " In another more specific embodiment, the hematopoietic cells are CD34 + CD38"CD33" CD 117 CD25 CD36".

In another embodiment, the hematopoietic cells are CD45 +. In another specific embodiment, the hematopoietic cells are CD34 + CD45 +. In another embodiment, the hematopoietic cell is Thy-1 +. In a specific embodiment, the hematopoietic cell is CD34 + Thy-1 +. In another embodiment, the hematopoietic cells are CD133 +. In specific embodiments, the hematopoietic cells are CD34 + CD133 + or CD133 + Thy-1 \ In another specific embodiment, the CD34 + hematopoietic cells are CXCR4 +. In another specific modality, the CD34 + hematopoietic cells are CXCR4. "In another embodiment, the hematopoietic cells are positive for KDR (vascular growth factor receptor 2.) In specific modalities, the hematopoietic cells are CD34 + KDR +, CD133 + KDR or Thy-1 + KDR + In certain other modalities, the hematopoietic cells are positive for aldehyde dehydrogenase (ALDH +), for example, the cells are CD34 + ALDH \ In certain other embodiments, CD34 + cells are CD45. "In specific embodiments, CD34 + cells, eg, CD34 +, CD45 cells" express one or more, or all, of miRNAs hsa-miR-380, hsa-miR -512, hsa-miR-517, hsa-miR-518c, hsa-mR-519b, and / or hsa-miR-520a.

In certain embodiments, the hematopoietic cells are CD34. Hematopoietic cells may also lack certain markers that indicate lineage involvement, or a lack of developmental ingenuity. For example, in another modality, the hematopoietic cells are HLA-DR. "In specific modalities, the hematopoietic cells are CD34 + HLA-DR", CD133 + HLA-DR ", Thy-1 + HLA-DR" or ALDH + HLA -DR. "In another embodiment, the hematopoietic cells are negative for one or more, preferably all, of the lineage markers CD2, CD3, CD11c, CD14c, CD14, CD16, CD19, CD24, CD56b, and glycophorin A.

In this manner, hematopoietic cells can be selected for use in the methods described herein on the basis of the presence of markers indicating an undifferentiated state, or on the basis of the absence of lineage markers indicating that at least one occurrence has occurred. some lineage differentiation. Methods of isolating cells, including hematopoietic cells, on the basis of the presence or absence of specific markers are discussed in detail, for example, in section 6.1.2, below.

The hematopoietic cells used in the methods provided herein may be a substantially homogeneous population, for example, a population comprising at least about 95%, at least about 98% or at least about 99% of a single hematopoietic cell. tissue source, or a population comprising hematopoietic cells that exhibit the same cellular markers associated with hematopoietic cells. For example, in various embodiments, the hematopoietic cells may comprise at least about 95%, 98% or 99% of hematopoietic cells of bone marrow, cord blood, placental blood, peripheral blood, or placenta, for example, perfused with placenta.

The hematopoietic cells used in the methods provided herein can be obtained from a single individual, for example, from a single placenta, or from a plurality of individuals, for example, they can be grouped. Where the hematopoietic cells are obtained from a plurality of individuals and grouped, the hematopoietic cells can be obtained from the same source of tissue. Thus, in various embodiments, the grouped hematopoietic cells are all placental, for example, placental perfused, all peripheral blood, all umbilical cord blood, all peripheral blood, and the like.

The hematopoietic cells used in the methods described herein, in certain embodiments, may comprise hematopoietic cells from two or more tissue sources. For example, in certain embodiments, when hematopoietic cells from two or more sources are combined for use in the methods herein, a plurality of the hematopoietic cells used to produce TSNK cells comprise placental hematopoietic cells, for example, placental perfusate. In several embodiments, the hematopoietic cells used to produce TSNK cells comprise hematopoietic cells from placenta and cord blood; of placenta and peripheral blood; of placenta and peripheral blood; or placenta and bone marrow. In a preferred embodiment, the hematopoietic cells comprise placental perfusate hematopoietic cells in combination with cord blood hematopoietic cells, wherein cord blood and placenta are from the same individual, i.e., where perfusate and blood are matched. cord. In embodiments wherein the hematopoietic cells comprise hematopoietic cells from two tissue sources, the hematopoietic cells of the sources can be combined in a ratio of, for example, 1:10, 2: 9, 3: 8, 4: 7, 5 : 6, 6: 5, 7: 4, 8: 3, 9: 2, 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3 , 1: 2, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1 or 9: 1. 6. 1.1 Hematopoietic Placental Stem Cells In certain embodiments, the hematopoietic cells used in the methods provided herein are placental hematopoietic cells. As used herein, "placental hematopoietic cells" means hematopoietic cells obtained from the placenta itself, and not from placental blood or umbilical cord blood. In one embodiment, the placental hematopoietic cells are CD34 +. In a specific embodiment, the placental hematopoietic cells are predominantly (eg, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) CD34 + CD38 cells. "In another specific embodiment, placental hematopoietic cells are predominantly (eg, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%). %, 90%, 95% or 98%) CD34 + CD38 + cells Placental hematopoietic cells can be obtained from a postpartum mammalian placenta (eg, human) by any means known to those skilled in the art, by example, by perfusion.

In another embodiment, the placental hematopoietic cell is CD45. "In a specific modality, the hematopoietic cell is CD34 + CD45." In another specific embodiment, the placental hematopoietic cells are CD34 + CD45 +. 6. 2. Production of Natural Killer Cells The production of NK cells by the present method comprises expanding a population of hematopoietic cells. During cell expansion, a plurality of hematopoietic cells within the hematopoietic cell population differentiate into NK cells.

In one embodiment, a method of producing a population of activated natural killer (NK) cells is provided, comprising: (a) seeding a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 (IL) - 15) and, optionally, one or more of the stem cell factor (SCF) and interleukin-7 (IL-7), wherein said optional IL-15 and SCF and IL-7 are not included within an undefined component of said medium, so that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.

In another embodiment, the NK cells provided herein are produced by a two-step process of NK cell expansion / differentiation and maturation. The first and second steps involve growing the cells in medium with a unique combination of cellular factors. In certain embodiments, the process involves (a) culturing and expanding a population of hematopoietic cells in a first medium, wherein a plurality of hematopoietic stem or progenitor cells within the population of hematopoietic cells differentiate into NK cells; and (b) expanding the NK cells of step (a) in a second medium, wherein the NK cells expand further and differentiate, and where the NK cells are matured (eg, activated or otherwise possessing cytotoxic activity) . In certain embodiments, the method includes any intermediate step between step (a) and step (b), no additional culture step before step (a), and / or no additional step (eg, maturation step) after the step (b). 6. 2.1. First Growing Step In certain embodiments, the methods provided herein comprise a first step of culturing and expanding a population of hematopoietic cells in a first medium, wherein a plurality of hematopoietic stem or progenitor cells within the population of hematopoietic cells differentiate into NK cells. .

Without being limited by any parameter, mechanism or theory, culture of the hematopoietic cells as provided herein results in continuous expansion of the hematopoietic cells and differentiation of NK cells from said cells. In certain embodiments, the hematopoietic cells, e.g., stem cells or progenitor cells, used in the methods provided herein expand and differentiate in the first step using a feeder layer. In other embodiments, the hematopoietic cells, e.g., stem cells or progenitor cells, expand and differentiate in the first step without the use of a feeder layer.

The independent expansion and differentiation of hematopoietic cell feeder cells can occur in any container compatible with culture and cell expansion, for example, bottle, tube, beaker, dish, multi-well plate, bag or the like. In a specific embodiment, the independent expansion of the hematopoietic cell feeder cell occurs in a bag, for example, a flexible, gas-permeable fluorocarbon culture bag (for example, from American Fluoroseal). In a specific embodiment, the container in which the hematopoietic cells expand is suitable for delivery, for example, to a site such as a hospital or military zone where the expanded NK cells expand and further differentiate.

In certain embodiments, the hematopoietic cells expand and differentiate, for example, in a continuous mode, in a first culture medium. In one embodiment, the first culture medium is a free medium of animal component. Exemplary animal component free media useful in the methods provided herein include, but are not limited to, Eagle Basal Medium (BME), Dulbecco's Modified Eagle Medium (DMEM), Glasgow Minimum Essential Medium (GMEM), Medium modified Dulbecco's Eagle / F-12 Ham nutrient mixture (DMEM / F-12), minimum essential medium (E), Iscove's modified Dulbecco's medium (IMDM), Ham's F-10 nutrient mixture (F-10 Ham), mixture of F-12 Ham nutrient (Ham F-12), RPMI-1640 medium, Wílliam medium E, STEMSPAN® (Cat. Stem Cell Technologies, Vancouver, Canada), basal growth medium Glycostem Basal Growth Medium (GBGM®), AIM-V® media (Invitrogen), X-VIVO ™ 10 (Lonza), X-VIVO ™ 15 (Lonza), OPTIMIZER (Invitrogen), STEMSPAN® H3000 (STEMCELL Technologies), CELLGRO COMPLETE ™ (Mediatech), or any modified variant or combinations thereof.

In preferred embodiments, the first culture medium comprises one or more medium supplements (e.g., nutrients, cytokines and / or factors). Suitable medium supplements for use in the methods provided herein include, for example, without limitation, serum such as human serum AB, fetal bovine serum (FBS) or fetal calf serum (FCS), vitamins, bovine serum albumin ( BSA), amino acids (e.g., L-glutamine), fatty acids (e.g., oleic acid, linoleic acid or palmitic acid), insulin (e.g., recombinant human insulin), transferin (human saturated iron transferrin), β- mercaptoethanol, stem cell factor (SCF), FMS tyrosine kinase 3 ligand (FU3-L), cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin -15 (IL-15), thrombopoietin (Tpo), heparin, or O-acetyl-carnitine (also referred to as acetylcarnitine, O-acetyl-L-carnitine or OAC). In a specific embodiment, the medium used comprises human serum AB. In another specific embodiment, the medium used herein comprises FBS. In another specific embodiment, the medium used herein comprises OAC.

In certain embodiments, the first medium does not comprise one or more of, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), protein 1 a macrophage inflammatory (MIP1a), or leukemia inhibitory factor (LIF).

Thus, in one aspect, a two-step method of producing NK cells is provided herein, wherein said first step comprises expanding and differentiating a population of hematopoietic cells in a first culture medium in the absence of feeder cells, in wherein a plurality of hematopoietic cells within said population of hematopoietic cells differentiate into NK cells during said expansion, and wherein the medium comprises SCF at a concentration of about 1 to about 150 ng / mL, IL-2 at a concentration of about 50 to about 1500 IU / mL, IL-7 at a concentration of about 1 to about 150 ng / mL, IL-15 at a concentration of about 1 to about 150 ng / mL and heparin at a concentration of about from 0.1 to about 30 lU / mL, and where said SCF, IL-2, IL-7, IL-15 and heparin are not comprised within an undefined component of said medium (e.g., serum). In certain embodiments, said medium comprises one or more of O-acetyl-carnitine (also referred to as acetylcarnitine, O-acetyl-L-carnitine or OAC), or a compound that affects the acetyl-CoA cycle in mitodronia, thiazo-vivine, and -27632, piintegrin, Rho kinase inhibitors (ROCK), caspase inhibitors or other anti-apoptotic compounds / peptides, NOVA-RS (Sheffield Bio-Science) or other small molecule growth enhancers. In certain modalitiessaid medium comprises nicotinamide. In certain embodiments, said medium comprises approximately 0.5 mM-10 mM OAC. In one embodiment, said medium comprises Stemspan® H3000, and / or DM MS: F 12 and approximately 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM OAC. In a specific embodiment of the method, said medium is GBGM®. In another specific embodiment, said medium comprises Stemspan® H3000 and approximately 5 mM OAC. In another specific embodiment, said medium comprises DMEM: F12 and approximately 5 nM OAC. The OAC may be added at any time during the culture methods provided herein. In certain modalities, said OAC is added to the first means on day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 of the culture. In a specific embodiment, said OAC is added to the first medium on day 7 of the first culture step. In a more specific embodiment, said OAC is added to the first medium on day 7 of the culture and is present in the first and second cultivation steps. In certain embodiments, said OAC is added to the second medium and / or during the second culture step. In some embodiments, said OAC is added to the second medium on day 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 of the culture.

In another specific embodiment, said medium is IMDM supplemented with approximately 5-20% BSA, approximately 1-10 μg / mL recombinant human insulin, approximately 10-50 μg / mL human iron transferrin saturated and approximately 10-50 μ? β-mercaptoethanol. In another specific embodiment, said medium does not comprise one or more, or none, of IL-11, IL-3, homeobox-B4 (HoxB4), and / or methylcellulose.

In other specific embodiments, said medium comprises SCF at a concentration of from about 0.1 to about 500 ng / mL; from about 5 to about 100 ng / mL; or approximately 20 ng / mL. In other specific embodiments, said medium comprises IL-2 at a concentration of from about 10 to about 2000 lU / mL; or from about 100 to about 500 IU / mL; or approximately 200 lU / mL. In other specific embodiments, said medium comprises IL-7 at a concentration of from about 0.1 to about 500 ng / mL; from about 5 to about 100 ng / mL; or approximately 20 ng / mL. In other specific embodiments, said medium comprises IL-15 at a concentration of from about 0.1 to about 500 ng / mL; from about 5 to about 100 ng / mL; or approximately 10 ng / mL. In other specific embodiments, said medium comprises heparin at a concentration of about 0.05 to about 100 IU / mL; or from about 0.5 to about 20 U / ml; or approximately 1.5 U / mL.

Still in another specific embodiment of the method, said medium further comprises Fms-type tyrosine kinase 3 ligand (Flt-3L) at a concentration of from about 1 to about 150 ng / mL, thrombopoietin (Tpo) at a concentration of about 1. at about 150 ng / mL, or a combination of about 0.1 to about 500 ng / mL; from about 5 to about 100 ng / mL; or approximately 20 ng / mL. In other specific embodiments, said medium comprises TPO at a concentration of from about 0.1 to about 500 ng / mL; from about 5 to about 100 ng / mL; or approximately 20 ng / mL.

In a more specific mode of the method, the first culture medium is GBGM®, which comprises approximately 20 ng / mL SCF, approximately 20 ng / mL IL-7, approximately 10 ng / mL IL-15. In another more specific embodiment of the method, the first culture medium is GBGM®, which comprises approximately 20 ng / mL SCF, approximately 20 ng / mL FH3-L, approximately 200 lU / mL IL-2, approximately 20 ng / mL IL -7, approximately 10 ng / mL IL-15, approximately 20 ng / mL Tpo, and approximately 1.5 U / mL heparin. In another specific embodiment, said first culture medium further comprises 10% human serum (e.g., human serum AB) or fetal serum (e.g., FBS).

In another embodiment, the hematopoietic cells are expanded by culturing said cells, for example, in said first medium, in contact with an immunomodulatory compound, for example, a TNF-α inhibitor compound, for a time and in an amount sufficient to cause a detectable increase in the proliferation of hematopoietic cells over a given time, compared to an equivalent number of hematopoietic cells not contacted with the immunomodulatory compound. See, for example, patent application publication of E.U.A. No. 2003/0235909, the description of which is incorporated herein by reference in its entirety. In certain embodiments, the immunomodulatory compound is an amino-substituted isoindoline. In a preferred embodiment, the immunomodulatory compound is 3- (4-amino-1-oxo-1,3-dihydro-isoindol-2-yl) -piperidine-2,6-dione; 3- (4'-aminoisoindolin-1'-one) -1-piperidine-2,6-dione; 4- (amino) -2- (2,6-dioxy (3-piperidyl)) - isoindoline-1,3-dione; or 4-amino-2- (2,6-dioxopiperidin-3-yl) isoindol-1,3-dione. In another preferred embodiment, the immunomodulatory compound is pomalidomide or lenalidomide. In another embodiment, said immunomodulatory compound is a compound having the structure wherein one of X and Y is C = 0, the other of X and Y is C = 0 or CH2, and R2 is hydrogen or lower alkyl, or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate or mixture of stereoisomers thereof. In another embodiment, said immunomodulatory compound is a compound having the structure where one of X and Y is C = 0 and the other is CH2 or C = 0; R is H, (d-C8) alkyl, (C3-C7) cycloalkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, benzyl, aryl, (C0-C4) alkyl -heterocycloalkyl (d-Ce), (C0-C4) alkyl -heteroaryl (C2-C5), C (0) R3, C (S) R3, C (0) OR4, alkyl (d -C8) -N (R6) 2, alkyl (d-C8) ) -OR5, (Ci-C8) alkyl -C (0) OR5, C (0) NHR3, C (S) NHR3, C (0) NR3R3 ', C (S) NR3R3' or alkyl (d-C8) - 0 (CO) R5; R2 is H, F, benzyl, alkyl (d-C8), alkenyl (C2-C8), or alkynyl (C2-C8); R3 and R3 are independently alkyl (d-C8), cycloalkyl (C3-C7), alkenyl (C2-C8), alkynyl (C2-C8), benzyl, aryl, alkyl (C0-d) -heterocycloalkyl (C ^ -d) ), alkyl (C0-C) -heteroaryl (C2-C5), (C0-C8) alkyl -N (R6) 2, alkyl (d-C8) -OR5, alkyl (d-C8) -C (0) OR5 , alkyl (d-C8) -0 (CO) R5 or C (0) OR5; R4 is alkyl (C ^ -C ^), alkenyl (C2-C8), alkynyl (C2-C8), alkyl (C-C4) -OR5, benzyl, aryl, (C0-C4) alkyl -heterocycloalkyl (Ci-Ce) ) or (C0-C4) alkyl-heteroaryl (C2-C5); R5 is (Ci-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, benzyl, aryl or (C2-C5) heteroaryl; each occurrence of R6 is independently H, (Ci-C8) alkyl, (C2-C8) alkenyl, (C2-C8) alkynyl, benzyl, aryl, (C2-C5) heteroaryl or (C0-C8) alkyl-C (O ) O-R5 or the R6 groups can be combined to form a heterocycloalkyl group; n is 0 or 1; Y * represents a chiral carbon center; or a salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. In another embodiment, said immunomodulator compound is a compound having the structure where: one of X and Y is C = 0 and the other is CH2 or C = 0; R is H or CH2OCOR '; (i) each of R1, R2, R3 or R4, independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R1, R2, R3 or R4 is nitro or -NHR5 and the remainder of R \ R2, R3 or R4 are hydrogen; R5 is hydrogen or alkyl of 1 to 8 carbon atoms R6 is hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro or fluoro; R 'is R7-CHR10-N (R8R9); R7 is m-phenylene or p-phenylene or - (CnH2n) - wherein n has a value of 0 to 4; each of R8 and R9 taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R8 and R9 taken together are tetramethylene, pentamethylene, hexamethylene, or -CH2CH2X1CH2CH2- wherein X1 is -O-, -S- or -NH-; R10 is hydrogen, alkyl of 8 carbon atoms, or phenyl; Y * represents a chiral carbon center; or a salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In a specific embodiment, the expansion of the hematopoietic cells is performed in IMDM supplemented with 20% BITS (bovine serum albumin, recombinant human insulin and transferin), SCF, ligand Flt-3, IL-3 and 4- (amino) - 2- (2,5-dioxo (3-piperidyl)) - isoindoline-1,3-dione (10 μ ?? in 0.05% DMSO). In a more specific embodiment, approximately 5 x 10 7 hematopoietic cells, eg, CD34 + cells, expand in the medium from about 5 x 10 10 cells to about 5 x 10 12 cells, which are resuspended in 100 ml_ of IMDM to produce a population of expanded hematopoietic cells. The population of expanded hematopoietic cells is preferably cryopreserved to facilitate shipping.

In several specific modalities, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% of the hematopoietic cells they differentiate to NK cells.

In certain embodiments, the method of expansion and differentiation of hematopoietic cells, as described herein, comprises maintaining the cell population comprising said hematopoietic cells at between about 2 x 10 4 and about 2 x 10 5 cells per milliliter during the expansion and differentiation. In certain other embodiments, the method of expansion and differentiation of hematopoietic cells, as described herein, comprises maintaining the cell population comprising said hematopoietic cells at no more than about 1 x 10 5 cells per milliliter.

The time for expansion and differentiation of hematopoietic cells into NK cells can be, for example, from about 3 days to about 120 days. In one embodiment, the differentiation time is around 7 days to about 75 days. In another modality, the differentiation time is around 14 days to approximately 50 days. In a specific modality, the differentiation time is around 21 days to about 28 days. 6. 2.2. Second step In the method provided herein, the hematopoietic cells, e.g., stem cells or progenitor cells, and natural killer cells, which result from the first step, expand further and differentiate in a second step, e.g., without the use of a layer. feeder or in the presence of feeder cells. The culture of the cells as provided herein results in continuous expansion, differentiation as well as maturation of the NK cells of the first step. In the second step, the NK cells are expanded, differentiated and matured, in a continuous mode, in a second culture medium, for example, comprising different cytokines and / or bioactive molecules than said first medium. In certain embodiments, the second culture medium is a free medium of animal component. Exemplary animal component free cell culture media are described in section 6.2.1, above.

Thus, in one aspect, a method of producing NK cells is provided herein, comprising expanding the NK cells of the first step, described above, into a second medium in the presence of feeder cells and in contact with interleukin-2 (IL -2). In specific embodiments, said second medium comprises cell growth medium comprising IL-2, for example, 10 IU / mL at 1000 IU / mL, and one or more of: human serum (e.g., human AB serum), bovine serum fetal (FBS) or fetal calf serum (FCS), for example, 5% - 15% FCS v / v; transferin, for example, 10 μ? / mL at 50 μ? / mL; insulin, for example, 5 μg |. { ? L · a 20 μ? /? T ?? -; ethanolamine, for example, 5 x 10"to 5 x 10" 5 M; oleic acid, for example, 0.1 μ? /? t ?? at 5 μ? /? t ?? -; linoleic acid, for example, 0.1 μg / μl to 5 μl / mL ·: palmitic acid, for example, 0.05 μg / G. at 2 μ? / ??? -; bovine serum albumin (BSA), for example, 1 μ9 /? to 5 μ9 / ??; and / or phytohaemagglutinin, for example, 0.01 μ9 / λ "_ to 1 μ9 /?" - In a more specific embodiment, said second medium comprises cell growth medium comprising FBS or FCS, eg, 10% FCS v / v, IL-2, transferin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin (BSA) and phytohemagglutinin In a more specific embodiment, said second medium comprises modified Dulbecco's medium from Iscove (IMDM), 10% FBS or FCS, 400 IU IL-2, 35 μg / mL transferin, 5 μg / mL insulin, 2 x 10 ~ 5 M ethanolamine, 1 μ? / ??? - oleic acid, 1 μ? / mL linoleic acid (Sigma-Aldrich), 0.2 μgglL · palmitic acid (Sigma-Aldrich), 2.5 μg / mL BSA (Sigma-Aldrich) and 0.1 μ9 / (? _ phytohemagglutinin.

In certain embodiments, the second medium does not comprise one or more of, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6), protein 1 a macrophage inflammatory (MIP1 a), or leukemia inhibitory factor (LIF).

In addition to the method, any of the means described above as compositions are provided herein.

Feeding cells, when used, of various cell types can be established. Examples of these cell types include, without limitation, fibroblasts, stem cells (e.g., placental stem cells adhering to tissue culture), fibroblasts (e.g., peripheral blood mononuclear cells (PBMC)), and cancer cells (e.g. , chronic myelogenous leukemia (CML) cells such as K562). In a specific embodiment, said culture in said second means comprises cultivating using feeder cells, for example, K562 cells and / or peripheral blood mononuclear cells (PBMCs), for example, at the time when cells initiate in said second medium, , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days later. In certain embodiments, the feeder cells optionally are of a different species as the cells they support. For example, human NK cells can be supported by mouse embryonic fibroblasts (primary culture or a telomerized line).

In certain embodiments, the feeder cells are optionally inactivated by irradiation (e.g., irradiation?) Or treatment with an anti-mitotic agent such as mitomycin C, to prevent them from overtaking the cells they are supporting, but allow the synthesis of important factors that support NK cells. For example, cells can be irradiated at a dose to inhibit proliferation but allow synthesis of important factors that They support human embryonic stem cells (hES) (approximately 4000 rads gamma irradiation).

The culture of NK cells for the second step can occur in any container compatible with culture and cell expansion, for example, bottle, tube, beaker, dish, multi-well plate, bag or the like. In a specific embodiment, the NK cell feeder-dependent culture occurs in a bag, eg, gas-permeable fluorocarbon flexible culture bag (e.g., from American Fluoroseal). In a specific embodiment, the container in which the NK cells are cultured is suitable for shipping, for example, to a site such as a hospital or military zone where the expanded NK cells expand more, differentiate and mature.

The differentiation of cells from step 1 into TSNK cells can be evaluated by detecting specific NK cell markers, for example, by flow cytometry. Specific NK cell markers include, but are not limited to, CD56, CD94, CD117 and NKp46. Differentiation can also be assessed by the morphological characteristics of NK cells, for example, large size, high protein synthesis activity in the abundant endoplasmic reticulum (ER) and / or preformed granules.

The time for expansion and differentiation of cells from step 1 into TSNK cells can be, for example, from about 3 days to about 120 days. In one embodiment, the differentiation time is around 7 days to about 75 days.

In another modality, the differentiation time is around 14 days to approximately 50 days. In a specific modality, the differentiation time is around 10 days to about 21 days.

The differentiation of hematopoietic cells into NK cells can be evaluated by detecting markers, for example, CD56, CD94, CD117, NKG2D, DNAM-1 and NKp46, for example, by flow cytometry. Differentiation can also be assessed by the morphological characteristics of NK cells, for example, large size, high protein synthesis activity in the abundant endoplasmic reticulum (ER) and / or preformed granules. Maturation of NK cells (e.g., TSNK cells) can be assessed by detecting one or more functionally relevant markers, eg, CD94, CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR, and the NKG2 family of activating receptors (for example, NKG2D). The maturation of NK cells (e.g., TSNK cells) can also be assessed by detecting specific markers during different stages of development. For example, in one embodiment, the pro-NK cells are CD34 +, CD45RA \ CD10 +, CD117 + and / or CD161. "In another embodiment, the immature cells are CD34 +, CD45RA \ CD10", CD117 + and / or CD161" In another embodiment, the immature NK cells are CD34-, CD117 \ CD161 \ NKp46"and / or CD94 / N KG2A. In another embodiment, the CD56brNlante NK cells are CD117 +, NKp46 +, CD94 / NKG2A +, CD16"and / or KIR + '\ In another embodiment, the CD56 cells, where NK are CD117", NKp46 +, CD94 / N KG2 A + / ", CD16 + and / or KIR +.

In a specific embodiment, the maturation of NK cells (e.g., TSNK cells) is determined by the percentage of NK cells (e.g., TSNK cells) which are CD161", CD94 + and / or NKp46 \ In a more specific embodiment, by at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of mature NK cells (e.g., TSNK cells) are NKp46 +. other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (e.g., TSNK cells) are CD94 +. specific, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of mature NK cells (e.g., TSNK cells) are CD161. " In certain embodiments, the differentiation of hematopoietic cells into NK cells is evaluated by detecting the level of expression of, for example, CD3, CD7 or CD127, CD10, CD14, CD15, CD16, CD33, CD34, CD56, CD94, CD117, CD161. , NKp44, NKp46, NKG2D, DNAM-1, 2B4 or TO-PRO-3, using, for example, antibodies to one or more of these cellular markers. Such antibodies can be conjugated to a detectable label, for example, as a fluorescent label, for example, FITC, R-PE, PerCP, PerCP-Cy5.5, APC, APC-Cy7 or APC-H7. 6. 3. Isolation of TSNK Cells Methods of isolating natural killer cells are known in the art and can be used to isolate TSNK cells. Natural killer cells can be isolated or enriched by staining cells from a tissue source, for example, peripheral blood, with antibodies to CD56 and CD3, and selecting for CD56 + CD3 cells. "TSNK cells can be isolated using commercially available equipment. for example, the NK cell isolation kit (Miltenyi Biotec) .The TSNK cells can also be isolated or enriched by removing different cells from the NK cells in a population of cells comprising the TSNK cells. TSNK can be isolated or enriched by omission of cells displaying non-NK cell markers, eg, antibodies to one or more of CD3, CD4, CD14, CD19, CD20, CD36, CD66, CD123, H LA DR and / or CD235a (Glycophorin A) Negative isolation can be carried out using commercially available equipment, for example, the NK cell negative isolation equipment (Dynal Biotech) .The cells isolated by these methods can be isolated. n additionally classify, for example, to separate CD16 + and CD16 cells ".

Cell separation can be achieved, for example, by flow cytometry, fluorescence activated cell sorting (FACS), or, preferably, magnetic cell sorting using conjugated microspheres with specific antibodies. The cells can be isolated, for example, using a magnetic activated classification (MACS) technique, a method for separating particles based on their ability to join magnetic spheres (e.g., about 0.5-100 μ? T? Diameter) comprising one or more specific antibodies, for example, anti-CD56 antibodies. Magnetic cell separation can be performed and automated using, for example, an AUTOMACS ™ separator (Miltenyi). A variety of useful modifications can be made to the magnetic microspheres, including covalent addition of antibody that specifically recognizes a particular cell surface molecule or hapten. The spheres are then mixed with the cells to allow binding. The cells then pass through a magnetic field to separate cells having the specific cell surface marker. In one embodiment, these cells can then be isolated and re-mixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells then pass once more through a magnetic field, isolating cells that bind both antibodies. Said cells can then be diluted in separate plates, such as microtiter plates for clonal isolation. 6. 4. Placenta perfusate TSNK cells from hematopoietic cells, e.g., stem or hematopoietic progenitors can be produced from any source, e.g., placental tissue, placental perfusate, umbilical cord blood, placental blood, peripheral blood, spleen, liver, or the like. In certain embodiments, hematopoietic stem cells are combined hematopoietic stem cells from placental perfusate and cord blood from the same placenta used to generalize placental perfusate. The placental perfusate comprising placental perfusate cells obtainable, for example, by the methods described in the U.S. Patents. Nos. 7,045,148 and 7,468,276, the descriptions of which are incorporated herein by reference in their totals. 6. 4.1. Composition of Cell Collection Peripheral perfusate and perfusate cells, from which stem cells or hematopoietic progenitors can be isolated, or useful in tumor suppression or treatment of an individual having tumor cells, cancer or a viral infection, for example, in combination with TSNK cells, as provided herein, can be harvested by perfusion from a mammal, e.g., human postpartum placenta using a placental cell harvesting composition. Perfusate of the placenta can be collected by perfusion of the placenta with any physiologically acceptable solution, for example, a saline solution, culture medium, or a more complex cellular collection composition. A cellular harvesting composition suitable for perfusing a placenta, and for the collection and storage of perfusate cells is described in detail in the application publication of E.U.A. No. 2007/0190042, which is incorporated herein by reference in its entirety.

The cell harvesting composition may comprise any physiologically acceptable solution suitable for the collection and / or culture of stem cells, for example, a saline solution (eg, phosphate buffered saline, Kreb's solution, modified Kreb's solution, Eagle, 0.9% NaCl, etc.), a culture medium (e.g., DMEM, H.DMEM, etc.), and the like.

The cellular harvesting composition may comprise one or more components that tend to retain placental cells, ie, prevent placental cells from drying, or delay the death of placental cells, reduce the number of placental cells in a population of cells that die, or the like, from the moment of harvesting to the moment of cultivation. Such components may be, for example, an apoptosis inhibitor (eg, a caspase inhibitor or JNK inhibitor); a vasodilator (eg, magnesium sulfate, an anti-hypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin, or magnesium sulfate, an inhibitor of phosphod iesterase, etc.); a necrosis inhibitor (e.g., 2- (1 H-indol-3-yl) -3-pentylamino-maleimide, pyrrolidine dithiocarbamate, or clonazepam); a TNF-a inhibitor; and / or an oxygen carrying perfluorocarbon (eg, perfluorooctyl bromide, perfluorodecyl bromide, etc.).

The cell harvesting composition may comprise one or more tissue degrading enzymes, for example, a metalloprotease, a serine protease, a neutral protease, a hyaluronidase, a RNase, or a DNase, or the like. Such enzymes include, but are not limited to, collagenases (for example, collagenase I, II, III or IV, a collagenase from Clostridium histolyticum, etc.); dispase, thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The cellular harvesting composition may comprise a bacteriocidally or bacteriostatically effective amount of an antibiotic. In certain non-limiting embodiments, the antibiotic is a macrolide (e.g., tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime, cefprozil, cefaclor, cefixime, or cefadroxil), a clarithromycin, an erythromycin, a penicillin (e.g. , penicillin V) or a quinolone (for example, ofloxacin, ciprofloxacin or norf loxacin), a tetracycline, a streptomycin, etc. In a particular embodiment, the antibiotic is active against Gram (+) and / or Gram (-) bacteria, for example, Pseudomonas aeruginosa, Staphylococcus aureus, and the like.

The cell harvesting composition may also comprise one or more of the following compounds: adenosine (from about 1 mM to about 50 mM); D-glucose (from about 20 mM to about 100 mM); Magnesium ions (from about 1 mM to about 50 mM); a macromolecule of molecular weight greater than 20,000 daltons, in one embodiment, present in an amount sufficient to maintain endothelial integrity and cell viability (e.g., a colloid that occurs synthetically or naturally, a polysaccharide such as dextran or a polyethylene glycol present in about 25 g / l to about 100 g / l, or from about 40 g / l to about 60 g / l); an antioxidant (for example, butylated hydroxyanisole, butylated hydroxytoluene, glutathione, vitamin C or vitamin E present from about 0.1 M to about 5 mM); an agent that prevents entry of calcium into cells (for example, verapamil present from about 2 μ? to about 25 μ?); nitroglycerin (for example, from about 0.05 g / L to about 0.2 g / L); an anticoagulant, in one embodiment, present in an amount sufficient to help prevent coagulation of residual blood (eg, heparin or hirudin present at a concentration of about 1000 units / I to approximately 100,000 units / l); or a compound containing amiloride (for example, amiloride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present from about 1.0 μ to about 5 μ). 6.4.2. Collection and Management of Placenta In general, a human placenta is recovered shortly after its expulsion after birth. In a preferred embodiment, the placenta is retrieved from a patient after informed consent and after taking a complete medical history of the patient and is associated with the placenta. Preferably, the medical history continues after delivery.

Before recovering the perfusate, umbilical cord blood and placental blood are eliminated. In certain modalities, after delivery, the cord blood in the placenta is recovered. The placenta can be subjected to a conventional cord blood recovery process. Typically a needle or cannula is used, with the aid of gravity, to exsanguinate the placenta (see, for example, Anderson, U.S. Patent No. 5,372,581, Hessel et al., U.S. Patent No. 5,415,665). The needle or cannula is usually placed in the umbilical vein and the placenta can be gently massaged to help drain cord blood from the placenta. Said cord blood recovery can be performed commercially, for example, LifeBank, Inc., Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell. Preferably, the placenta is drained by gravity without further manipulation to minimize tissue disruption during cord blood recovery.

Typically, a placenta is transported from the delivery room or delivery to another location, for example, a laboratory, for cord blood recovery and perfusate collection. The placenta is preferably transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-28 ° C), for example, by placing the placenta, with a nearby umbilical cord attached, in a zip-lock plastic bag sterile, which is then placed in an insulated container. In another embodiment, the placenta is transported in a cord blood collection equipment substantially as described in the U.S. patent. No. 7,147,626. Preferably, the placenta is delivered to the laboratory four to twenty-four hours after delivery. In certain embodiments, the proximal umbilical cord is fastened, preferably within 4-5 cm (centimeter) of the placental disc insertion prior to retrieval of cord blood. In other modalities, the proximal umbilical cord is held after retrieval of cord blood but before further processing of the placenta.

The placenta, before collection of the perfusate, can be stored under sterile conditions and at room temperature or at a temperature of 5 to 25 ° C (centigrade). The placenta can be stored for a longer period of forty-eight hours, and preferably for a period of four to twenty-four hours before perfusing the placenta to remove any residual cord blood. The placenta is preferably stored in an anticoagulant solution at a temperature of 5 ° C to 25 ° C (Centigrade). Suitable anticoagulant solutions are well known in the art. For example, a solution of heparin or sodium warfarin may be used. In a preferred embodiment, the anticoagulant solution comprises a heparin solution (eg, 1% w / w in 1: 1000 solution). The exsanguinated placenta is preferably stored for no more than 36 hours before collecting placental perfusate. 6. 4.3. Placenta perfusion Methods of perfusing mammalian placentas and obtaining placental perfusate are described, for example, in Hariri, U.S. Patents. Nos. 7,045,148 and 7,255,879, and in the application publications of E.U.A. Nos. 2007/0190042 and 20070275362, the descriptions of which are incorporated herein by reference in their entireties.

Perfusate solution can be obtained perfused by, for example, saline solution, culture medium or cell harvesting compositions described above, via placental vasculature. In one embodiment, a mammalian placenta is perfused by passage of perfusion solution through one or both of the umbilical artery and umbilical vein. The flow of perfusion solution through the placenta can be achieved using, for example, gravity flow in the placenta.

Preferably, the perfusion solution is forced through the placenta using a pump, for example, a peristaltic pump. The umbilical vein, for example, can be cannulated with a cannula, for example, a TEFLON® or plastic cannula, which is connected to a sterile connecting apparatus, such as a sterile tube. The sterile connecting apparatus is connected to a perfusion manifold.

In preparation for perfusion, the placenta is preferably oriented in such a way that the umbilical artery and umbilical vein are located at the highest point of the placenta. The placenta can be perfused by a perfusion solution through the placental vasculature, or through the vasculature of the placenta and surrounding tissue. In one embodiment, the umbilical artery and the umbilical vein are simultaneously connected to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The perfusion solution passes into the vein and umbilical artery. The perfusion solution exudes from and / or passes through the walls of the blood vessels from the surface of the placenta that was fixed to the mother's uterus during pregnancy. The perfusion solution can also be introduced through the opening of the umbilical cord and allowed to flow or percolate out of the openings in the wall of the placenta which are in interface with the maternal uterine wall. In another modality, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or passed through the umbilical artery and collected from the umbilical veins, that is, passed through only the placental vasculature (fetal tissue).

In one embodiment, for example, the umbilical artery and the umbilical vein are connected simultaneously, for example, to a pipette that is connected via a flexible connector to a reservoir of the perfusion solution. The perfusion solution is passed into the umbilical vein and artery. The perfusion solution exudes from and / or passes through the walls of the blood vessels in the surrounding tissues of the placenta, and is collected in a suitable open container from the surface of the placenta that was fixed to the mother's uterus during gestation The perfusion solution can also be introduced through the umbilical cord opening and allowed to flow or percolate out of the openings in the wall of the placenta which are in interface with the maternal uterine wall. Placental cells that are harvested by this method, can be referred to as a "pan" method, are typically in a mixture of fetal and maternal cells.

In another modality, the perfusion solution is passed through the umbilical veins and collected from the umbilical artery, or passed through the umbilical artery and collected from the umbilical veins. Placental cells collected by this method, which can be referred to as a "closed circuit" method, are typically almost exclusively fetal.

The closed circuit perfusion method, in one modality, can be performed in the following manner. A postpartum placenta is obtained within approximately 48 hours after birth. The umbilical cord is held and cut above the forceps. The umbilical cord can be discarded, or it can be processed to retrieve, for example, umbilical cord stem cells, and / or to process the umbilical cord membrane for the production of a biomaterial. The amniotic membrane can be retained during perfusion, or it can be separated from the chorion, for example, using direct dissection with the fingers. If the amniotic membrane is separated from the chorion before perfusion, it can, for example, be discarded or processed, for example, to obtain stem cells by enzymatic digestion, or to produce, for example, an amniotic membrane biomaterial, for example, the biomaterial described in the US application publication No. 2004/0048796. After cleaning the placenta of all visible blood clots and residual blood, for example, using sterile gauze, the umbilical cord vessels are exposed, for example, by partially cutting the umbilical cord membrane to expose a cord cross section. The vessels are identified, and open, for example, by advancing a closed crocodile clip across the cut end of each vessel. The apparatus, for example, plastic tube connected to an infusion device or peristaltic Itica pump, is then inserted into each of the placental arteries. The pump can be any pump suitable for the purpose, for example, a peristaltic pump. The plastic tube, connected to a sterile collection container, for example, a blood bag such as a 250 ml collection bag, is then inserted into the placenta vein. Alternatively, the tube connected to the pump is inserted into the placenta vein, and tubes to a collection reservoir (s) are inserted into one or both of the placental arteries. The placenta is then perfused with a volume of perfusion solution, for example, approximately 750 ml of perfusion solution. Cells are then collected in the perfusate, for example, by centrifugation.

In one embodiment, the proximal umbilical cord is held during the perfusion, and more preferably, is held within 4-5 cm (centimeters) of the insertion of the cord in the placental disc.

The first collection of perfusion fluid from a mammalian placenta during the exsanguination process is usually colored with residual red blood cells from cord blood and / or placental blood. The perfusion fluid becomes more colorless as the perfusion proceeds and the residual bead globules are washed out of the placenta. In general, 30 to 100 mL of perfusion fluid is adequate to wash blood from the placenta at the beginning, but more or less perfusion fluid can be used depending on the results observed.

The volume of perfusion fluid used to perfuse the placenta may vary depending on the number of placental cells to be harvested, the size of the placenta, the number of collections to be made from a single placenta, etc. In various embodiments, the volume of perfusion fluid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is perfused with 700-800 mL of perfusion fluid after exsanguination.

The placenta can be perfused a plurality of times over the course of several hours or several days. When the placenta is to be perfused a plurality of times, it can be maintained or cultured under aseptic conditions in a container or other suitable container, and perfused with a cellular harvesting composition, or a standard perfusion solution (e.g., saline solution). normal such as phosphate buffered saline ("PBS") with or without an anticoagulant (eg, heparin, sodium warfarin, coumarin, bíshydroxycoumarin) and / or with or without an antimicrobial agent (eg, β-mercaptoethanol (0.1 mM), antibiotics such as streptomycin (for example, 40-100 μg ml), penicillin (for example, at 40U / ml), amphotericin B (for example, at 0.5μ? / ???). Isolated placenta is maintained or cultured for a period of time without collecting the perfusate, so that the placenta is maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or 2 or 3 0 more days before perfusion and perfusate collection. The perfused placenta can be maintained for one or more additional time (s), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, for example, 700-800 mL perfusion fluid. The Placenta can be perfused 1, 2, 3, 4, 5 or more times, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment, perfusion of the placenta and collection of perfusion solution, eg, placental cell harvesting composition, is repeated until the number of nucleated cells recovered falls below 100 cells / ml. Perfusates at different time points can be processed more individually to recover cell-dependent populations of time, e.g., total nucleated cells. You can also group perfused different time points. 6. 4.4. Perfusate of Placenta and Cells Placenta Perfusate Typically, the placental perfusate from a single placental perfusion comprises from about 100 million to about 500 million nucleated cells, including hematopoietic cells from which TSNK cells can be produced by the method described herein. In certain embodiments, the placental cells or perfusate cells comprise CD34 + cells, e.g., stem cells or hematopoietic progenitors. Said cells can, in a more specific embodiment, comprise CD34 + CD45"progenitor or stem cells, CD34 + CD45 + stem or progenitor cells, or the like In certain embodiments, the perfusate or perfusate cells are cryopreserved before the isolation of hematopoietic cells. In certain other embodiments, the placental perfusate comprises, or the perfusate cells comprise, only fetal cells, or a combination of fetal cells and maternal cells. 6. 5. TSNK cells In one aspect, TSNK cells are provided herein, the NK cells produced by the methods described herein (eg, two-step method). Furthermore, a population of cells comprising the TSNK cells produced by the methods described herein (eg, two-step method) is provided herein. In a specific embodiment, said NK cells (e.g., TSNK cells) are CD3"CD56 + CD16". In another specific embodiment, said NK cells (e.g., TSNK cells) are additionally CD94 + CD117 +. In another specific embodiment, said NK cells (e.g., TSNK cells) are additionally CD161. "In another specific embodiment, said NK cells (e.g., TSNK cells) are additionally NKG2D +. In another specific embodiment, said NK cells are additionally NKp46 + In another specific embodiment, said NK cells are additionally CD226 +.

In certain modalities, more than 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98% of said TSNK cells are CD56 + and CD16 \ In other modalities, at least 50% , 60%, 70%, 80%, 82%, 84%, 86%, 88% or 90% of said TSNK cells are CD3"and CD56 + In other embodiments, at least 50%, 52%, 54%, 56%, 58% or 60% of said TSNK cells are NKG2D In other modalities, less than 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of said cells are NKB1 + In certain other modalities, less 30%, 20%, 10%, 8%, 6%, 4% or 2% of said TSNK cells are NKAT2 + In certain other modalities, less than 30%, 20%, 10%, 8%, 6% , 4% or 2% of said TSNK cells are CD56"and CD16 +. In more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65% or 70% of said TSNK CD3 \ CD56 + cells are NKp46 + . In other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85% of said TSNK CD3", CD56 + cells are CD117 \ In other more specific modalities, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of said TSNK CD3 cells", CD56 + are CD94 +. In other more specific embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of said TSNK CD3", CD56 * cells are CD161". In other more specific modalities, at least 10%, 12%, 14%, 16%, 18% or 20% of said TSNK CD3"cells, CD56 + are CD226 +, in more specific modalities, at least 20%, 25% , 30%, 35% or 40% of said TSNK CD3 \ CD56 + cells are CD7 + In more specific modalities, at least 30%, 35%, 40%, 45%, 50%, 55% or 60% of said cells TSNK CD3", CD56 + are CD5 +.

In several other embodiments, TSNK cells can be combined with, for example, NK cells, wherein said NK cells have been isolated from a tissue source and have not expanded; NK cells isolated from a tissue source and expanded, or NK cells produced by a different method, for example, natural killer cells CD56 + CD16 +, for example, in ratios of, for example, about 1:10, 2: 9 3: 8, 4: 7, 5: 6, 6: 5, 7: 4, 8: 3, 9: 2, 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1 : 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1 or approximately 9: 1. As used in this context, "isolated" means that the cells have been removed from their normal tissue environment.

TSNK cells may have a fetal genotype or a maternal genotype. For example, since the postpartum placenta, as a source of suitable hematopoietic cells to produce TSNK cells, comprises tissue and cells from the fetus and from the mother, the placental perfusate may comprise only fetal cells, or a substantial majority of cells fetal (eg, more than about 90%, 95%, 98% or 99%) or may comprise a mixture of fetal and maternal cells (eg, fetal cells comprise less than about 90%, 80%, 70%, 60% or 50% of the total nucleated cells of the perfusate). In one embodiment, TSNK cells are derived only from hematopoietic cells from fetal placenta, eg, cells obtained from closed-circuit perfusion of the placenta wherein the perfusate produces perfusate comprising a substantial majority, or only, hematopoietic cells of fetal placenta. In another embodiment, TSNK cells are derived from fetal and maternal cells, for example, cells obtained by perfusion by the pan method (see above), wherein perfusion produced perfusate comprising a mixture of fetal and maternal placental cells. Thus, in one embodiment, a population of intermediate natural killer cells derived from the placenta, the substantial majority of which have the fetal genotype, is provided herein. In another embodiment, a population of intermediate natural killer cells derived from placenta are provided which comprise natural killer cells that have the fetal genotype and natural killer cells that have the maternal phenotype.

Also provided herein are populations of TSNK cells comprising natural killer cells not produced by the methods described herein. For example, in one embodiment, a population of TSNK cells that also comprises natural killer cells isolated from, for example, umbilical cord blood, peripheral blood, bone marrow, or a combination of two or more of the above is provided. , or NK cells expanded by a method different from the methods described herein. Said populations of TSNK cells may comprise TSNK cells and other NK cells in, for example, a ratio of approximately 1:10, 2: 9, 3: 8, 4: 7, 5: 6, 6: 5, 7: 4 8: 3, 9: 2, 10: 1, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2 : 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 100: 1, 95: 5, 90:10, 85:15, 80:20 , 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15 : 85, 10:90, 5:95, 100: 1, 95: 1, 90: 1, 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1 50: 1, 45: 1, 40: 1, 35: 1, 30: 1, 25: 1, 20: 1, 15: 1, 10: 1, 5: 1, 1: 1, 1: 5, 1 : 10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70 , 1:75, 1:80, 1:85, 1:90, 1:95 or about 1: 100 or similar.

In certain embodiments, isolated natural killer cells (e.g., TSNK cells) or populations enriched for natural killer cells (e.g., TSNK cells) can be evaluated by detecting one or more functionally relevant markers, e.g., CD94, CD161, NKp44, DNAM-1, 2B4, NKp46, CD94, KIR and the NKG2 family of activating receptors (e.g., NKG2D). In some embodiments, the purity of isolated or enriched natural killer cells can be confirmed by detecting one or more of CD56, CD3 and CD16.

Optionally, the cytotoxic activity of isolated or enriched natural killer cells can be evaluated, for example, in a cytotoxicity assay using tumor cells, for example, cultured tumor cells K562, LN-18, U937, WER1-RB-1, U- 118MG, HT-29, HCC2218, KG-1 or U266, or the like as target cells. 6. 6. TSNK Cells in Combination with Placental Perfusate Also herein are provided compositions comprising TSNK cells in combination with placental perfusate, placental perfusate cells and / or adherent placental cells, for example, for use in suppressing the proliferation of a tumor cell or plurality of tumor cells. 6. 6.1. Combinations of TSNK and Perfused Cells or Perfusion Cells Also herein are provided compositions comprising combinations of TSNK cells and placental perfusate and / or placental perfusate cells. In one embodiment, for example, a placental perfusate volume supplemented with TSNK cells is provided herein. In specific embodiments, for example, each milliliter of placental perfusate is supplemented with approximately 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more TSNK cells. In another embodiment, placental perfusate cells are supplemented with TSNK cells. In certain other embodiments, when placental perfusate cells are combined with TSNK cells, the placental perfusate cells in general comprise about, more than about, or less than about, 50%, 45%, 40%, 35%, %, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells. In certain other embodiments, when TSNK cells are combined with a plurality of placental perfusate cells and / or combined natural killer cells, the NK cells in general comprise about, more than about, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total number of cells. In certain other embodiments, when TSNK cells are used to supplement placental perfusate, the volume of solution (eg, saline, culture medium or the like) in which the cells are suspended comprises about, more than about, or less than about , 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or 1% of the total volume of perfusate plus cells, wherein the TSNK cells are suspended at about 1 x 104, 5 x 10 x 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more cells per milliliter before supplementation.

In other embodiments, any of the above combinations of cells, in turn, is combined with umbilical cord blood or umbilical cord blood nucleated cells.

In addition, placenta perfusate is obtained here which is obtained from two or more sources, for example, two or more placentas, and are combined, for example, grouped. Said pooled perfusate may comprise approximately equal volumes of perfusate from each source, or may comprise volumes different from each source. The relative volumes of each source can be selected randomly, or based on, for example, a concentration or amount of one or more cellular factors, eg, cytokines, growth factors, hormones or the like; the number of placental cells perfused from each source; or other perfused characteristics of each source. The perfusate of multiple infusions of the same placenta can be grouped similarly.

Similarly, placental perfusate cells, and intermediate natural killer cells derived from placenta are provided herein, which are obtained from two or more sources, eg, two or more placentas, or are pooled. Said pooled cells may comprise approximately equal numbers of cells from two or more sources, or different numbers of cells from one or more of the pooled sources. The relative numbers of cells of each source can be selected based on, for example, the number of one or more specific cell types in the cells to be grouped, for example, the number of CD34 + cells, etc.

Also herein are provided TSNK cells, and combos of TSNK cells with placental perfusate and / or placental perfusate cells, which have been tested to determine the extent or amount of tumor suppression (i.e., potency) to be expected from, for example, a given number of TSNK cells, or a given volume of perfusate. For example, an aliquot or cell sample number is contacted with a known number of tumor cells under conditions where the tumor cells would otherwise proliferate, and the proliferation rate of the tumor cells in the presence of placental perfusate, cells of perfusate, and the proliferation rate of tumor cells in the presence of perfusate of placenta, perfusate cells, natural killer cells of placenta, or combinations thereof, over time (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks, or more) is compared to the proliferation of an equivalent number of tumor cells in the absence of perfusate, perfusate cells, natural killer cells of placenta, or combinations thereof. The potency of the cells can be expressed, for example, as the number of cells or volume of solution required to suppress tumor cell growth, for example, by approximately 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, or similar.

In certain modalities, TSNK cells are provided as administrable units of pharmaceutical grade. Said units can be supplied in discrete volumes, for example, 15 ml_, 20 ml_, 25 ml, 30 ml, 35 ml_, 40 ml, 45 ml_, 50 ml_, 55 ml_, 60 ml_, 65 ml_, 70 ml, 75 ml , 80 mL, 85 mL, 90 mL, 95 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, 500 mL, or the like. Such units can be provided to contain a specified number of cells, for example, TSNK cells alone, or TSNK cells in combination with other NK cells or perfusate cells, for example, 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more cells per unit. In specific embodiments, the units may comprise approximately, at least approximately, or at most approximately 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107 , 1 x 108, 5 x 108 or more cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108 , 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more cells per unit. Said units may be provided to contain specified numbers of TSNK cells, and / or any other cell.

In the above embodiments, TSNK cells or combinations of TSNK cells with other NK cells, perfusate or perfusate cells can be autologous to a recipient (i.e., obtained from the container), or allogeneic to a recipient (i.e., obtained from at least one other individual from said vessel).

In certain embodiments, each unit of cells is marked to specify one or more of volume, number of cells, type of cells, whether the unit has been enriched by a particular type of cell, and / or power of a given number of cells. cells in a unit, or a given number of milliliters of the unit, that is, if the cells in the unit cause a measurable suppression of proliferation of a particular type or types of tumor cell. 6. 6.2. Combinations of TSNK Cells and Cells Adherent Placenta Mother In other embodiments, TSNK cells, either alone or in combination with placental perfusate or placental perfusate cells, are supplemented with isolated adherent placental cells, e.g., placental stem cells and multipotent placental cells as described, for example, in the US patents Nos. De Hariri 7,045,148 and 7,255,879, and in the patent application publication of E.U.A. Not 2007/0275362, the descriptions of which are incorporated herein by reference in their totals. "Adherent placental cells" means that the cells are adherent to a tissue culture surface, eg, tissue culture plastic. Adherent placental cells useful in the compositions and methods described herein are not trophoblasts, embryonic germ cells or embryonic stem cells. In certain embodiments, adherent placental stem cells are used as feeder cells during the processes (eg, two-step method) as described above.

TSNK cells, either alone or in combination with placental perfusate or placental perfusate cells can be supplemented with, for example, 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5? 108, or more adherent placental cells per milliliter, or 1 x 10 5 x 1 O 4, 1 x 105, 5 x 105, 1 x 1 O 6, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more cells per milliliter, or 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more adherent placental cells. Adherent placental cells in the combinations can be, for example, adherent placental cells that have been cultured for, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 , 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 population doublings, or more.

Isolated adherent placental cells, when cultured in primary cultures or expanded in cell culture, adhere to the tissue culture substrate, eg, tissue culture container surface (eg, tissue culture plastic). The adherent placental cells in culture assume a starry appearance usually fibroblast, with a number of cytoplasmic processes that extend from the central cell body. However, adherent placental cells morphologically distinguishable from fibroblasts cultured under the same conditions, such as adherent placental cells exhibit a greater number of such processes than fibroblasts. Morphologically, the adherent placental cells are also distinguishable from hematopoietic stem cells, which usually assume a more rounded, or cobbled, morphology in culture.

Isolated adherent placental cells, and adherent placental cell populations, useful in the compositions and methods provided herein, express a plurality of markers that can be used to identify and / or isolate cells, or populations of cells comprising adherent placental cells. Adherent placental cells, and adherent placental cell populations useful in the compositions and methods provided herein include adherent placental cells and cell populations containing adherent placental cells obtained directly from the placenta, or any part thereof ( for example, amnion, chorion, amnion-chorion plaque, placenta cotyledons, umbilical cord, and the like). The adherent placental stem cell population, in one embodiment, is a population (i.e., two or more) of adherent placental stem cells in culture, eg, a population in a container, e.g., a bag.

Adherent placental cells generally express the markers CD73, CD105, and CD200, and / or OCT-4, and do not express CD34, CD38 or CD45. Adherent placental stem cells can also express HLA-ABC (MHC-1) and HLA-DR. These markers can be used to identify adherent placental cells, and to distinguish adherent placental cells from other cell types. Since adherent placental cells can express CD73 and CD105, they can have mesenchymal stem cell type characteristics. The lack of expression of CD34, CD38 and / or CD45 identifies adherent placental stem cells as non-hematopoietic stem cells.

In certain embodiments, the isolated adherent placental cells described herein suppress detectably cancer cell proliferation or tumor growth.

In certain embodiments, the isolated adherent placental cells are isolated placental stem cells. In certain other embodiments, isolated adherent placental cells are multipotent isolated placental cells. In a specific embodiment, the isolated adherent placental cells are CD34", CD10 + and CD105 + as detected by flow cytometry In a more specific embodiment, isolated adherent placental cells CD34", CD1CT, CD105 + are placental stem cells . In another more specific embodiment, the isolated placental cells CD34", CD10 +, CD105 + are multipotent adherent placental cells.In another specific embodiment, isolated placental cells CD34", CD10 +, CD105 + have the potential to differentiate into cells of a phenotype neutral, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype. In a more specific embodiment, the adherent placenta cells isolated CD34", CD10 +, CD105 * are additionally CD200 + In another more specific embodiment, the isolated adherent placenta cells CD34", CD10 +, CD105 + are additionally CD90 + or CD45" , as detected by flow cytometry In another more specific embodiment, isolated adherent placenta cells CD34", CD10 +, CD105 + are additionally CD90 + or CD45", as detected by flow cytometry. adherent placental cells CD34", CD10 +, CD105 +, CD200 + are additionally CD90 + or CD45", as detected by flow cytometry In another more specific modality, adherent placental cells CD34", CD10 +, CD105 +, CD200 + they are additionally CD90 * and CD45", as detected by flow cytometry. In another more specific embodiment, adherent placental cells CD34", CD10 +, CD105", CD200 \ CD90 +, CD45" are additionally CD80"and CD 86", as detected by flow cytometry.

In one embodiment, isolated adherent placental cells are CD200 +, HLA-G +. In a specific embodiment, said isolated adherent placental cells are also CD73 + and CD105 +. In another specific embodiment, said isolated adherent placental cells are also CD34", CD38" or CD45. "In a more specific embodiment, said isolated adherent placental cells are also CD34", CD38", CD45", CD73 + and CD105 +. In another embodiment, said isolated adherent placental cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies.

In another embodiment, the isolated adherent placental cells are CD73 +, CD105 +, CD200. In a specific embodiment of said populations, said isolated adherent placental cells are also HLA-G +. In another specific embodiment, said isolated adherent placental cells are also CD34", CD38" or CD45. "In another specific embodiment, said isolated adherent placental cells are also CD34", CD38 'and CD45. "In a more specific embodiment, said isolated adherent placental cells are also CD34", CD38", CD45"and HLA-G \ In another specific embodiment, said isolated adherent placental cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of bodies. of embryoid type.

In another embodiment, isolated adherent placental cells are CD200 +, OCT-4 +. In a specific embodiment, said isolated adherent placental cells are also CD73 + and CD105 +. In another specific embodiment, said isolated adherent placental cells are also HLA-G +. In another specific embodiment, said isolated adherent placental cells are also CD34 ', CD38"and CD45". In a more specific embodiment, said isolated adherent placental cells are also CD34", CD38", CD45", CD73" CD105 + and HLA-G +. "In another specific embodiment, the isolated adherent placental cells also produce one or more bodies of embryoid type when grown under conditions that allow the formation of embryoid-like bodies.

In another embodiment, isolated adherent placental cells are CD73 +, CD105 + and HLA-G +. In a specific embodiment, isolated adherent placental cells are also CD34", CD38" or CD45. "In another specific embodiment, said isolated adherent stem cells are also OCT-4 + In another specific embodiment, said adherent stem cells are also CD200 * In a modality more specific, said adherent stem cells are also CD34", CD38", CD45 \ OCT-4 + and CD200 +.

In another embodiment, isolated adherent placental cells are CD73 +, CD105 + stem cells, wherein said cells produce one or more embryoid-like bodies under conditions that allow the formation of embryoid-like bodies. In a specific embodiment, said isolated adherent placental cells are also CD34", CD38" or CD45. "In another specific embodiment, the isolated adherent placental cells are also CD34", CD38"and CD45". In another specific embodiment, isolated adherent placental cells are also OCT-4 +. In a more specific embodiment, said isolated adherent placental cells are also OCT-4 *, CD34", CD38" and CD45 In another embodiment, the isolated adherent placental cells are OCT-4 + stem cells, wherein said isolated adherent placental cells produce one or more embryoid-like bodies when cultured under conditions that allow the formation of embryoid-like bodies, and wherein said stem cells have been identified as detectably suppressing cancer cell proliferation or tumor growth.

In various modalities, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 %, at least 90% or at least 95% of said isolated adherent placental cells are OCT-4 +. In a specific embodiment of the above populations, said isolated adherent placental cells are also CD73 + and CD105 +. In another specific embodiment, said isolated adherent placental cells are also CD34", CD38" or CD45. "In another specific embodiment, said stem cells are CD200.In a more specific embodiment, said isolated adherent placental cells are also CD73. CD105 +, CD200 +, CD43", CD38" and CD45". In another specific embodiment, said isolated adherent placental cells have expanded, for example, passed at least once, at least three times, at least five times, at least 10 times, at least 15 times or by at least 20 times.

In a more specific embodiment of any of the foregoing embodiments, isolated adherent placental cells express ABC-p (a placental-specific ABC transport protein, see, eg, Allikmets et al., Cancer Res. 58 (23): 5337 -9 (1998)).

In another embodiment, isolated adherent placental cells CD29 \ CD44 \ CD73 \ CD90 +, CD105 +, CD200 +, CD34"and CD133 \ In another embodiment, the isolated adherent placental cells secrete constitutively IL-6, IL-8 and Monocyte chemoattractant protein (MCP-1).

Each of the isolated adherent placental cells referred to above may comprise cells obtained and isolated directly from a mammalian placenta., or cells that have been cultured and passed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30 or more times, or a combination of them. The tumor cell suppressor pluralities of the isolated adherent placental cells described above may comprise at least, or not more than, 1 x 104, 5 x 10 4, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or more 1 x 104, 5 x 104, 1 x 105 , 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011 or cells of placenta adherents isolated. 6. 6.3. Compositions that Include Media Conditioning of Adherent Placental Cells The use of a composition comprising TSNK cells and additionally conditioned medium is also provided herein, wherein said composition is tumor suppressor, or is effective in the treatment of cancer or viral infection. Adherent placental cells as described in section 6.6.2, above can be used to produce conditioned media that is tumor cell, anti-cancer or anti-viral cell suppressor, ie, medium comprising one or more secreted biomolecules or excreted by cells that have a detectable tumor cell suppressive effect, anti-cancer effect or antiviral effect. In various embodiments, the conditioned medium comprises medium in which the cells have proliferated (i.e., been cultured) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, the conditioned medium comprises medium wherein said cells have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90% confluency. Said conditioned medium can be used to support the culture of a separate population of cells, for example, placental cells, or cells of another class. In another embodiment, the conditioned medium provided herein comprises medium wherein the isolated adherent placental cells, for example, isolated adherent placental stem cells or isolated adherent placental multipotent cells, and cells different from the isolated adherent placental cells, for example, non placental stem cells or multipotent cells, have been cultured.

Said conditioned medium can be combined with any of, or any combination of TSNK cells, placental perfused, placental perfusate cells to form a composition that is tumor cell suppressor, anti-cancer or antiviral. In certain embodiments, the composition comprises less than half of conditioned media in volume, eg, about, or less than about, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% , 10%, 5%, 4%, 3%, 2% or 1% by volume.

Thus, in one embodiment, a composition comprising TSNK cells and culture medium is provided herein from a culture of isolated adherent placental cells, wherein said isolated adherent placental cells (a) are adhere to a substrate; and (b) are CD34, CD10 + and CD105 +; wherein said composition detectably suppresses the growth or proliferation of tumor cells, or is anti-cancer or antiviral. In a specific embodiment, the isolated adherent placental cells are CD34", CD10 + and CD105 +, as detected by flow cytometry In a more specific embodiment, the isolated adherent placenta cells CD34", CD10 +, CD105 + are stem cells from placenta. In another more specific embodiment, the isolated placental cells CD34 ', CD10 +, CD105 + are multipotent adherent placental cells. In another specific embodiment, the isolated placenta cells CD34 ', CD10 +, C105 + have the potential to differentiate cells of a neural phenotype, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype. In a more specific embodiment, the adherent placenta cells isolated CD34", CD10 +, CD105 + are additionally CD200 + In another more specific embodiment, the adherent placenta cells isolated CD34", CD10 +, CD105 + are additionally CD90 + or CD45"as detected by flow cytometry In another more specific modality, isolated adherent placenta cells CD34", CD10 +, CD105 +, CD200 + are additionally CD90 + or CD45", as detected by flow cytometry. adherent placental cells CD34", CD10 +, CD105 +, CD200 + are additionally CD90 + and CD45", as detected by flow cytometry In another more specific modality, adherent placental cells CD34", CD10 \ CD105 +, CD200 + , CD90 +, CD45"are additionally CD80" and CD86", as detected by flow cytometry.

In another embodiment, a composition comprising TSNK cells and culture medium is provided from a culture of isolated adherent placental cells, wherein said isolated adherent placental cells (a) adhere to a substrate; and (b) express CD200 and HLA-G, or express CD73, CD105 and CD200, or express CD200 and OCT-4, or express CD73, CD105 and HLA-G, or express CD73 and CD105 and facilitate the formation of one or more embryoid-like bodies in a population of placental cells comprising the placental stem cells when said population is cultured under conditions that allow the formation of embryoid-like bodies, or express OCT-4 and facilitate the formation of one or more bodies of embryoid type in a population of placental cells comprising the placental stem cells when said population is cultured under conditions that allow the formation of embryoid-like bodies; wherein said composition detectably suppresses the growth or proliferation of tumor cells, or is anti-cancer or antiviral. In a specific embodiment, the composition further comprises a plurality of said isolated adherent placental cells. In another specific embodiment, the composition comprises a plurality of non-placental cells. In a more specific embodiment, said non-placental cells comprise CD34 + cells, for example, hematopoietic progenitor cells, such as peripheral blood hematopoietic progenitor cells, cord blood hematopoietic progenitor cells, or hematopoietic blood progenitor cells of placenta. Non-placental cells can also comprise stem cells, such as mesenchymal stem cells, e.g., mesenchymal stem cells derived from bone marrow. Non-placental cells can also be one or more types of adult cells or cell lines. In another specific embodiment, the composition comprises an antiproliferative agent, for example, an a nti-M I P-1 a or anti-M I P-1β antibody.

In a specific embodiment, the culture medium conditioned by one of the cells or cell combinations described above is obtained from a plurality of isolated adherent placental cells co-cultured with a plurality of tumor cells from about 1: 1 to about 2: 1, approximately 3: 1, approximately 4: 1 or approximately 5: 1 isolated adherent placental cells to tumor cells. For example, the conditioned culture medium or supernatant can be obtained from a culture comprising about 1 × 10 5 isolated adherent placenta cells, about 1 × 10 6 isolated adherent placenta cells, about 1 × 10 7 isolated or approximately adherent placental cells. 1 x 108 adherent placental cells isolated, or more. In another specific embodiment, the conditioned culture medium or supernatant is obtained from a co-culture comprising from about 1 × 10 5 to about 5 × 10 5 isolated adherent placenta cells and about 1 × 10 5 tumor cells; from about 1 x 106 to about 5 x 106 isolated adherent placental cells and about 1 x 10 6 tumor cells; from about 1 x 107 to about 5 x 107 isolated adherent placental cells and about 1 x 107 tumor cells; or from about 1 x 108 to about 5 x 108 isolated adherent placental cells and about 1 x 10 8 tumor cells. 6. 7. Conservation of Cells Cells, e.g., TSNK cells or placental perfusate cells comprising stem cells or hematopoietic progenitor cells, can be preserved, i.e., placed under conditions that allow long-term storage, or under conditions that inhibit cell death, e.g. by apoptosis or necrosis.

Placental perfusate can be produced by passage of a cellular harvesting composition through at least part of the placenta, for example, through the placental vasculature. The cellular harvesting composition comprises one or more compounds that act to preserve cells contained within the perfusate. Said placental cell harvesting composition may comprise an apoptosis inhibitor, necrosis inhibitor and / or an oxygen carrier perfluorocarbide, as described in the application publication of E.U.A. No. 20070190042 related, the description of which is incorporated herein by reference in its entirety.

In one embodiment, the perfusate or a population of placental cells are harvested from a mammalian postpartum placenta, eg, human, by contacting the perfusate or population of cells with a cellular harvesting composition comprising an apoptosis inhibitor and a oxygen carrier perfluorocarbide, wherein said apoptosis inhibitor is present in an amount and for a time sufficient to reduce or prevent apoptosis in the placental cell population, for example, adherent placental cells, for example, placental stem cells or multipotent placental cells, compared to a population of cells not contacted with the apoptosis inhibitor. For example, the placenta can be perfused with the cellular harvesting composition, and placental cells, for example, total nucleated placental cells, are isolated therefrom. In a specific embodiment, the apoptosis inhibitor is a caspase inhibitor. In another specific embodiment, said inhibitor of apoptosis is a JNK inhibitor. In a more specific embodiment, said JNK inhibitor does not modulate the differentiation or proliferation of adherent placental cells, for example, adherent placental stem cells or adipose multipotent placental cells. In another embodiment, the cellular harvesting composition comprises said apoptosis inhibitor and said oxygen carrying perfluorocarbon in separate phases. In another embodiment, the cellular harvesting composition comprises said apoptosis inhibitor and said oxygen carrying perfluorocarbon in an emulsion. In another embodiment, the cellular harvest composition additionally comprises an emulsifier, for example, lecithin. In another embodiment, said apoptosis inhibitor and said perfluorocarbon are between about 0 ° C and about 25 ° C at the time of contacting the placental cells. In another more specific embodiment, said apoptosis inhibitor and said perfluorocarbon are between about 2 ° C and 10 ° C, or between about 2 ° C and about 5 ° C, at the time of contacting the placental cells. In another more specific embodiment, said contact is made during transport of said population of cells. In another more specific embodiment, said contact is made during the freezing and thawing of said population of cells.

In another embodiment, placental perfusate and / or placental perfusate cells can be harvested and preserved upon contacting the perfusate and / or cells with an apoptosis inhibitor and an organ preservative compound, wherein said apoptosis inhibitor is present in a amount and for a sufficient time to reduce or prevent apoptosis of cells, compared to perfused or placental cells not contacted with the apoptosis inhibitor. In a specific embodiment, the oxygen-conserving compound is UW solution (described in U.S. Patent No. 4,798,824, also known as VIASPAN ™, see also Soouthard et al., Transplantation 49 (2): 251-257 (1990) or solution described in Stern et al, U.S. Patent No. 5,552,267, the descriptions of which are incorporated herein by reference in their entireties In another embodiment, said organ-conserving composition is hydroxyethyl starch, lactobionic acid, raffinose or a combination thereof In another embodiment, the cellular harvesting composition of placenta further comprises an oxygen carrying perfluorocarbon, either in two phases or as an emulsion.

In another embodiment of the method, the placental cells are contacted with a cellular harvesting composition comprising an oxygen carrier perfluorocarbon inhibitor, organ preservative compound, or combination thereof, during perfusion. In another embodiment, the placental cells are contacted with said cell harvesting compound after collection by perfusion.

Typically, during collection, enrichment and cellular isolation of placenta, it is preferable to minimize or eliminate cellular stress due to hypoxia and mechanical stress. In another embodiment of the method, therefore, placental perfusate or a population of placental cells is exposed to a hypoxic condition during collection, enrichment or isolation for less than six hours during said storage, wherein a hypoxic condition is a concentration of oxygen that is less than the normal concentration of blood oxygen. In a more specific embodiment, said perfusate or population of placental cells is exposed to said hypoxic condition for less than two hours during said storage. In other more specific embodiments, said population of placental cells is exposed to said hypoxic condition for less than one hour, or less than thirty minutes, or is not exposed to a hypoxic condition, during collection, enrichment or isolation. In another specific embodiment, said population of placental cells is not exposed to shear stress during collection, enrichment or isolation. Cells, for example, placental perfusate cells, hematopoietic cells, e.g., CD34 + hematopoietic stem cells; NK cells, for example, TSNK cells; Isolated adherent placental cells provided herein may be cryopreserved, for example, in cryopreservation medium in small containers, for example, ampoules or septum vials. In certain embodiments, the cells provided herein are cryopreserved at a concentration of approximately 1 x 104-5 x 108 cells per mL. In more specific embodiments, the cells provided herein are cryopreserved at a concentration of about 1 × 10 4, 5 × 10 4, 1 × 10 5, 5 × 10 5, 1 × 10 6, 5 × 10 6, 1 × 107, 5 × 10 7 cells per mL.

Suitable cryopreservation medium includes, but is not limited to, normal saline, culture medium including, for example, growth medium, or cell freezing medium, for example, commercially available cell freezing medium, for example, C2695, C2639 or C6039 (Sigma); CryoStor® CS2, CryoStor® CS5 or CryoStor® CS10 (BioLife Solutions). The cryopreservation medium preferably comprises DMSO (dimethyl sulfoxide), at a concentration of, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% (v / v). The cryopreservation medium may comprise additional agents, for example, methylcellulose, dextran, albumin (for example, human serum albumin), trehalose, and / or glycerol. In certain embodiments, the cryopreservation medium comprises about 1% -10% DMSO, about 25% -75% dextran and / or about 20-60% human serum albumin (HSA). In certain embodiments, the cryopreservation medium comprises about 1% -10% DMSO, about 25% -75% trehalose and / or about 20-60% human HSA. In a specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% dextran and 40% HSA. In a more specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% dextran (10% w / v in normal saline) and 40% HSA. In another specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% trehalose and 40% HSA. In a more specific embodiment, the cryopreservation medium comprises 5% DMSO, 55% trehalose (10% w / v in normal saline) and 40% HSA. In another specific embodiment, the cryopreservation medium comprises CryoStor® CS5. In another specific embodiment, the cryopreservation medium comprises CryoStor® CS10.

The cells provided herein can be cryopreserved by any of a variety of methods, and at no stage of cell culture, expansion or differentiation. For example, the cells provided herein may be cryopreserved immediately after isolation of the tissues or organs of origin, for example, perfused placenta or umbilical cord blood, or during, or after, the first or second step of the delineated methods. before. In certain embodiments, the hematopoietic cells, e.g., hematopoietic stem or progenitor cells are cryopreserved within about 1, 5, 10, 15, 20, 30, 45 minutes or within about 1, 2, 4, 6, 10, 12 , 18, 20 or 24 hours after isolation from the tissues or organs of origin. In certain embodiments, said cells are cryopreserved within 1, 2 or 3 days after isolation of the tissues or organs of origin. In certain embodiments, said cells are cryopreserved after being cultured in a first medium as described in section 6.2.1 above, by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. In some embodiments, said cells are cryopreserved after being cultured in a first medium as described in section 6.2.1 above, by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25, 27 or 28, in a second medium by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days as described in section 6.2.1 above.

In one aspect, a method of cryopreservating a population of NK cells, e.g., TSNK cells, is provided herein. In one embodiment, said method comprises: (a) sowing a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-1 L (IL-15) and, optionally, one or more of the stem cell factor (SCF) and interleukin-7 (IL-7), wherein said optional IL-15 and SCF and IL-7 are not comprised within an undefined component of said medium, so that the population expands, and a plurality of stem cells or hematopoietic progenitors within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells, and (c) cryopreservating the NK cells of step (b) in a means of cryopreservation. In a specific embodiment, said step (c) further comprises (1) preparing a cell suspension solution; (2) adding cryopreservation medium to the cell suspension solution of step (1) to obtain cryopreserved cell suspension; (3) cooling the cryopreserved cell suspension of step (3) to obtain a cryopreserved sample; and (4) store the cryopreserved sample below -80 ° C. In certain embodiments, the method includes any intermediate step between step (a) and (b), and between step (b) and (c), and / or no additional culture step before step (a).

In another embodiment, said method of cryopreservating a population of NK cells, eg, TSNK cells comprises: (a) expanding a population of hematopoietic stem or progenitor cells into a first medium comprising one or more of the stem cell factor (SCF) , IL-2, interleukin-7 (IL-7), interleukin-15 (IL-15) and heparin, and wherein said SCF, IL-2, IL-7 and IL-15 are not comprised within a non-component. defined from said medium, and wherein a plurality of hematopoietic stem or progenitor cells within said population of hematopoietic stem or progenitor cells differentiate into NK cells during said expansion; (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells, and (c) cryopreservating the NK cells of step (b) in a means of cryopreservation. In a specific embodiment, said step (c) further comprises (1) preparing a cell suspension solution; (2) adding cryopreservation medium to the cell suspension solution of step (1) to obtain cryopreserved cell suspension; (3) cooling the cryopreserved cell suspension of step (3) to obtain a cryopreserved sample; and (4) classify the cryopreserved sample below -80 ° C. In certain embodiments, the method includes any intermediate step between step (a) and (b), and between step (b) and (c).

The cells provided herein are preferably cooled in a controlled rate freezer, for example, to about 0.1, 0.3, 0.5 or 1 ° C / min during the cryopreservation A preferred cryopreservation temperature is from about -80 ° C to about -180 ° C, preferably from about -125 ° C to about -140 ° C. The cryopreserved cells can be transferred to liquid nitrogen before thawing to be used. In some embodiments, for example, once the ampoules reach approximately -90 ° C, they are transferred to a liquid nitrogen storage area. The cryopreserved cells are preferably thawed at a temperature of about 25 ° C to about 40 ° C, preferably at a temperature of about 37 ° C. In certain embodiments, the cryopreserved cells are thawed after being cryopreserved for approximately 1, 2, 4, 6, 10, 12, 18, 20 or 24 hours, or for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. In certain embodiments, the cryopreserved cells are thawed after being cryopreserved for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 months. In certain embodiments, the cryopreserved cells are thawed after being cryopreserved for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

Suitable de-icing medium includes, but is not limited to, normal saline, plasma culture medium including, for example, growth medium, eg, RPMI medium. In preferred embodiments, the thawing medium comprises one or more medium supplements (e.g., nutrients, cytokines and / or factors). Supplements medium suitable for thawing cells provided herein include, for example, without limitation, serum such as human AB serum, fetal bovine serum (FBS) or fetal calf serum (FCS), vitamins, human serum albumin (HSA ), bovine serum albumin (BSA), amino acids (e.g., L-glutamine), fatty acids (e.g., oleic acid, linoleic acid or palmitic acid), insulin (e.g., recombinant human insulin), transferrin (human transferrin iron saturated), beta-mercaptoethanol, stem cell factor (SCF), ligand tyrosine kinase 3 Fms (FU3-L), cytokines such as interleukin-2 (IL - 2), interleukin-7 (IL-7 ), interleukin-15 (IL-15), thrombopoietin (Tpo) or heparin. In a specific embodiment, the thawing medium useful in the methods provided herein comprises RPMI. In another specific embodiment, said defrosting means comprises plasmalite. In another specific embodiment, said defrosting means comprises approximately 0.5-20% FBS. In another specific embodiment, said defrosting means comprises about 1, 2, 5, 10, 15 or 20% FBS. In another specific embodiment, said defrosting means comprises approximately 0.5-20% HSA. In another specific embodiment, said defrosting means comprises about 1, 2.5, 5, 10, 15 or 20% HSA. In a more specific embodiment, said defrosting means comprises RPMI and approximately 10% FBS. In another more specific embodiment, said means of thawing comprises plasmalite and about 5% HSA.

The methods of cryopreservation provided herein can be optimized to allow long-term storage, or under conditions that inhibit cell death, for example, by apoptosis and necrosis. In one embodiment, the post-thawing cells comprise more than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% of viable cells, as determined, for example, by Automatic cellular counter or tryptan blue method. In another embodiment, post-thawing cells comprise about 0.5, 1, 5, 10, 15, 20 or 25% of dead cells. In another embodiment, post-thawing cells comprise about 0.5, 1, 5, 10, 15, 20 or 25% of early apoptotic cells. In another modality, approximately 0.5, 1, 5, 10, 15 or 20% of post-thawing cells undergo apoptosis after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days after thawing, for example, as determined by an apoptosis test (for example, TO-PR03 or AnnV / PI Apoptosis assay kit). In certain embodiments, the post-thawing cells are re-cryopreserved after being cultured, expanded or differentiated using methods provided herein. 6. 8. Uses of TSNK Cells The TSNK cells provided herein are used in methods of treating individuals having cancer, for example, individuals having solid tumor cells and / or blood cells with cancer, or persons having a viral infection. The TSNK cells provided herein can also be used in methods of suppressing the proliferation of tumor cells. 6. 8.1. Treatment of Individuals with Cancer In one embodiment, a method of treating an individual having a cancer, for example, a blood cancer or a solid tumor, which comprises administering to said individual a therapeutically effective amount of TSNK cells is provided. In certain embodiments, the individual has a deficiency of natural killer cells, for example, a deficiency of NK cells active against the individual's cancer. In a specific modality, the method further comprises administering to said isolated perfusate of isolated placenta or isolated placental perfusate cells, for example, a therapeutically effective amount of placental perfusate or isolated placental perfusate cells. In another specific embodiment, the method further comprises administering to said subject an effective amount of an immunomodulatory compound, for example, an immunomodulatory compound. described in section 6.2.1, earlier, or thalidomide. As used herein, an "effective amount" is an amount that, for example, results in a detectable improvement of, reducing the progress of, or eliminating, one or more symptoms of a cancer from which the individual suffers.

In a specific embodiment, the cancer is blood cancer, for example, a leukemia or a lymphoma. In more specific modalities, the cancer is an acute leukemia, for example, acute T cell leukemia, acute myelogenous leukemia (AL), acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, B-precursor acute lymphoblastic leukemia, acute lymphoblastic leukemia of precursor T, Burkitt's leukemia (Burkitt's lymphoma), or acute biphenotypic leukemia; a chronic leukemia, for example, chronic myeloid lymphoma, chronic myelogenous leukemia (CML), chronic monocytic leukemia, chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma, or prolificcytic B-cell leukemia; hairy cell lymphoma; T-cell prolymphocytic leukemia; or a lymphoma (e.g., histiocytic lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, plasma cell neoplasm (e.g., plasma cell myeloma, plasmacytoma, an immunoglobulin deposition disease) monoclonal, or a heavy chain disease), extranodal marginal zone B-cell lymphoma (MALT lymphoma), nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma , large mediatinal (thymic) B-cell lymphoma, large intravascular B-cell lymphoma, primary effusion lymphoma, large granular T-cell lymphocytic leukemia, aggressive NK cell leukemia, adult T-cell leukemia / lymphoma, NK cell lymphoma / Extranodal T, nasal type, enteropathy type T cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, mycosis fungoides (Sezary syndrome) ), a primary cutaneous CD30-positive T cell lymphoproliferative disorder (eg, primary cutaneous anaplastic large cell lymphoma or lymphomatoid papulosis), angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma, unspecified, anaplastic large cell lymphoma, a Hodgkin's lymphoma or a predominant nodular lymphocyte Hodgkin's lymphoma. In another specific modality, the cancer is multiple myeloma or myelodysplastic syndrome.

In certain other specific embodiments, the cancer is a solid tumor, for example, a carcinoma, such as an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a carcinoma of the lung, a thyroid carcinoma, a nasopharynx carcinoma, a melanoma (for example, a malignant melanoma), a nonmelanoma skin carcinoma, or an unspecified carcinoma; a tumor unmolds; a small demoplastic round cell tumor; an endocrine tumor; an Ewing's sarcoma; a germ cell tumor (eg, testicular cancer, ovarian cancer, choriocarcinoma, endodermal sinus tumor, germinoma, etc.); a hepatosblastoma; a hepatocellular carcinoma; a neuroblastoma; a soft tissue sarcoma of no rhabdomyosarcoma; an osteosarcoma; a retinoblastoma; a rhabdomyosarcoma; or a Wilms tumor. In another embodiment, the solid tumor is pancreatic cancer or breast cancer. In other modalities, the solid tumor is an acoustic neuroma; an astrocytoma (for example, a grade I pilocytic astrocytoma, a grade II low grade astrocytoma, a grade III anaplastic astrocytoma, or a grade IV glioblastoma multiforme); a chordoma; a craniopharyngioma; a glioma (for example, a glioma of the brainstem, an ependymoma, a mixed glioma, an optic nerve glioma, or a subependymoma); a glioblastoma; a medulloblastoma; a meningioma; a metastatic brain tumor; an oligodendroglioma; a pineoblastoma; a pituitary tumor; a primitive neurorectodermal tumor; or a schwannoma. In another modality, cancer is prostate cancer.

In certain embodiments, the individual having a cancer, for example, a blood cancer or a solid tumor, for example, an individual who has a deficiency of natural killer cells, is an individual who has received a bone marrow transplant before said bone marrow transplant. administration. In certain modalities, the bone marrow transplant was in treatment of said cancer. In certain other modalities, the bone marrow transplant was under treatment for a condition other than said cancer. In certain embodiments, the individual received an immunosuppressant in addition to said bone marrow transplant. In certain embodiments, the individual who has had a bone marrow transplant exhibits one p plus symptoms of graft-versus-host disease (GVHD) at the time of said administration. In certain other embodiments, the individual who has had a bone marrow transplant is administered said cells before a symptom of graft-versus-host disease (GVHD) is manifested.

In certain specific embodiments, the individual having a cancer, e.g., a blood cancer, has received at least one dose of a TNFa inhibitor, e.g., ETANERCEPT® (Enbrel), prior to said administration. In specific embodiments, said individual received said dose of a TNFa inhibitor within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months of diagnosis of said cancer. In a specific embodiment, the individual who has received a dose of a TNFa inhibitor exhibits acute myeloid leukemia. In a more specific embodiment, the individual who has received a dose of a TNFa inhibitor and exhibits acute myeloid leukemia also exhibits omission of the long arm of chromosome 5 in globules. In another embodiment, the individual having a cancer, for example, a blood cancer, exhibits a Philadelphia chromosome.

In certain other embodiments, the cancer, for example, a blood cancer or a solid tumor, in said individual is refractory to one or more anti-cancer drugs. In a specific modality, the cancer is refractory to GLEEVEC® (imatinib mesylate).

In certain embodiments, the cancer, for example, a blood cancer, in said individual responds to at least one anti-cancer drug; in this embodiment, perfused placenta, isolated placental perfusate cells, isolated natural killer cells, e.g., natural placental killer cells, e.g., natural killer cells derived from placental, isolated combined natural killer cells, and / or combinations thereof, and optionally an immunomodulatory compound, are added as adjunctive treatments or as a combination therapy with said anti-cancer drug. In certain other embodiments, the individual having a cancer, for example, a blood cancer, has been treated with at least one anti-cancer drug, and has relapsed, prior to said administration.

In one aspect, herein is provided a method of treating an individual having multiple myeloma, comprising administering to the individual (1) lenalidomide; (2) melphalan; and (3) expanded NK cells, wherein said NK cells are effective to treat multiple myeloma in said individual. In a specific embodiment, said NK cells are NK cells of cord blood, or NK cells produced from cord blood hematopoietic cells, e.g., hematopoietic stem cells. In another embodiment, said NK cells have been produced by any of the methods described herein to produce NK cells, for example, to produce TSNK cells. In another embodiment, said NK cells have been expanded prior to said administration. In another embodiment, said lenalidomide, melphalan and / or NK cells are administered separately from each other. In certain specific embodiments of the method of treating an individual with multiple myeloma, said NK cells are produced by a method comprising: expanding a population of hematopoietic stem or progenitor cells into a first medium comprising one or more of the stem cell factor (SCF) ), IL-2, interleukin-7 (IL-7), interleukin-15 (IL-15) and heparin, and wherein said SCF, IL-2, IL-7 and IL-15 are not included within a component undefined of said medium, and wherein a plurality of stem cells or hematopoietic progenitors within said population of stem cells or hematopoietic progenitors differentiate into NK cells during said expansion; and expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2).

In another aspect, a method of treating an individual having chronic lymphocytic leukemia (CLL), which comprises administering to the individual a therapeutically effective dose of (1) lenalidomide, is provided; (2) melphalan; (3) Fludarabine; and (4) expanded NK cells, for example, TSNK cells, wherein said NK cells are cord blood NK cells, or NK cells produced from cord blood hematopoietic stem cells. In another embodiment, said NK cells have been produced by any of the methods described herein to produce NK cells, for example, to produce TSNK cells. In a specific embodiment of any of the above methods, said NK cells have been expanded for at least 10 days before said administration. In a specific embodiment of any of the above methods, said lenalidomide, melphalan, fludarabine and expanded NK cells are administered to said individual separately. In certain specific embodiments of the method of treating an individual with CLL, said NK cells are produced by a method comprising: expanding a population of stem cells or hematopoietic progenitors in a first medium comprising one or more of the stem cell factor (SCF) , IL-2, interleukin-7 (IL-7), interleukin-15 (IL-15) and heparin, and wherein said SCF, IL-2, IL-7 and IL-15 are not comprised within a non-component. defined from said medium, and wherein a plurality of stem cells or hematopoietic progenitors within said population of stem cells or hematopoietic progenitors differentiate into NK cells during said expansion; and expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce activated NK cells. 6. 8.2. Suppression of Tumor Cell Proliferation In addition, a method of suppressing the proliferation of tumor cells, which comprises contacting the tumor cells with TSNK cells, is provided herein. Optionally, the tumor cells and / or TSNK cells are contacted with isolated placental perfusate or isolated placental perfusate cells. In another specific embodiment, the tumor cells and / or TSNK cells are additionally contacted with an immunomodulatory compound, for example, an immunomodulatory compound described in section 6.2.1, above, or thalidomide, so that the proliferation of the tumor cells is reduces detectably compared to tumor cells of the same type not contacted with TSNK cells. Optionally, the tumor cells and / or TSNK cells contacted with an immunomodulatory compound are contacted with isolated placental perfusate or isolated placental perfusate cells.

As used herein, "contact" with respect to cells, in a modality encompasses direct physical contact, eg, cell-cell, between placental perfusate, placental perfusate cells, natural killer cells, eg, TSNK cells , and / or isolated combined natural killer cells and tumor cells. In another embodiment, "contact" encompasses presence in the same physical space, for example, perfused placenta, placental perfusate cells, natural killer cells, for example, natural killer cells intermediate of placenta, and / or combined natural killer cells isolated they are placed in the same container, for example, culture dish, multi-well plate) as tumor cells. In another embodiment, "contacting" perfusate of placenta, placental perfusate cells, combined natural killer cells or natural killer cells intermediate of placenta, and tumor cells is achieved, for example, by injecting or infusing the perfusate of placenta or cells, by example, placental perfusate cells, combined natural killer cells or natural killer cells, eg, natural killer cells intermediate placenta in an individual, eg, a human comprising tumor cells, eg, a cancer patient. "Contact" in the context of immunomodulatory compounds and / or thalidomide are physically contacted with each other, or are placed within the same physical volume (e.g., a cell culture container or an individual).

In a specific embodiment, the tumor cells are blood cancer cells, for example, leukemia cells or lymphoma cells. In more specific embodiments, the cancer is an acute leukemia, for example, acute T cell leukemia cells, acute myelogenous leukemia (AML) cells, acute promyelocytic leukemia cells, acute myeloblastic leukemia cells, acute megakaryoblastic leukemia cells B-precursor acute lymphoblastic leukemia cells, T-precursor acute lymphoblastic leukemia cells, Burkitt's leukemia cells (Burkitt's lymphoma), or acute biphenotypic leukemia cells; chronic leukemia cells, for example, chronic myeloid lymphoma cells, chronic myelogenous leukemia (CML) cells, chronic monocytic leukemia cells, chronic lymphocytic / small lymphocytic leukemia cells, or B-cell prolymphocytic leukemia cells; hairy cell lymphoma cells; T-cell prolymphocytic leukemia cells; or lymphoma cells, eg, histiocytic lymphoma cells, lymphoplasmacytic lymphoma cells (e.g., Waldenstrom macroglobulinemia cells), splenic marginal zone lymphoma cells, plasma cell neoplasm cells (e.g., myeloma cells) of plasma cell, plasmacytoma cells, monoclonal immunoglobulin deposition disease, or heavy chain disease), extranodal marginal B-cell lymphoma cells (MALT lymphoma), nodal marginal zone B-cell lymphoma cells (NMZL) , follicular lymphoma cells, mantle cell lymphoma cells, large intravascular B cell lymphoma cells, primary effusion lymphoma cells, large T-cell granular lymphocytic leukemia cells, aggressive NK cell leukemia cells, leukemia / adult T cell lymphoma, nasal-type cells - extranodal NK / T cell lymphoma, T-cell lymphoma cells enteropathy, hepatosplenic T cell lymphoma cells, blastic NK cell lymphoma cells, mycosis fungoides (Sezary syndrome), CD30-positive primary cutaneous T cell lymphoproliferative disorder cells (e.g., anaplastic large cell lymphoma) primary cutaneous or lymphomatoid papulosis), angioimmunoblastic T cell lymphoma cells, unspecified peripheral T cell lymphoma cells, nodular lymphocyte predominant Hodgkin lymphoma cells, or Hodgkin lymphoma cells. In another specific embodiment, the tumor cells are multiple myeloma cells or myelodysplastic syndrome cells.

In specific embodiments, the tumor cells are solid tumor cells, e.g., carcinoma cells, e.g., adenocarcinoma cells, adenocortical carcinoma cells, colon adenocarcinoma cells, colorectal adenocarcinoma cells, colorectal carcinoma cells, carcinoma cells. of ductal cell, lung carcinoma cells, thyroid carcinoma cells, nasopharyngeal carcinoma cells, melanoma cells (e.g., malignant melanoma cells), non-melanoma skin carcinoma cells, or unspecified carcinoma cells; tumor cells strips; small desmoplastic round cell tumor cells; endocrine tumor cells; Ewing's sarcoma cells; germ cell tumor cells (e.g., testicular cancer cells, ovarian cancer cells, choriocarcinoma cells, endodermal sinus tumor cells, germinoma cells, etc.); hepatosblastoma cells; hepatocellular carcinoma cells; neuroblastoma cells; soft tissue sarcoma cells from non-rhabdomyosarcoma; osteosarcoma cells; retinoblastoma cells; rhabdomyosarcoma cells; or Wilms tumor cells. In another embodiment, the tumor cells are pancreatic cancer cells or breast cancer cells. In other embodiments, the solid tumor cells are acoustic neuroma cells; astrocytoma cells (eg, grade I polycytic astrocytoma cells, grade II low grade astrocytoma cells, grade III anaplastic astrocytoma cells, or multiform glioblastoma grade IV); chordoma cells; craniofaringioma cells; glioma cells (e.g., brainstem glioma cells; ependymoma cells; mixed glioma cells; optic nerve glioma cells; or subependymoma cells); glioblastoma cells; medulloblastoma cells; meningioma cells; tumor cells of metastatic brain; oligodendroglioma cells; pineoblastoma cells; pituitary tumor cells; primitive neuroectodermal tumor cells; or schwannoma cells. In another embodiment, the tumor cells are prostate cancer cells.

As used herein, "therapeutically beneficial" and "therapeutic benefits" include, but are not limited to, for example, reduction in the size of a tumor; reducing or stopping the expansion of a tumor; reduction in the number of cancer cells in a tissue sample, eg, blood sample, per unit volume; the clinical improvement in any symptom of the particular cancer or tumor said individual has, the reduction or cessation of worsening of any particular cancer symptom the individual has, etc. 6. 8.3. Treatment of cancers using TSNK cells and other anti-cancer agents The treatment of an individual having cancer using the TSNK cells described herein may be part of an anti-cancer therapy regimen that includes one or more other anti-cancer agents. Such anti-cancer agents are well known in the art. Specific anti-cancer agents that can be administered to an individual having cancer, for example, an individual having tumor cells, in addition to the TSNK cells, and optionally perfused, perfusate cells, natural killer cells other than TSNK cells, include, but they are not limited to: acivicin; aclarubicin; benzoyl hydrochloride; Acronine; adozelesina; adrucil; aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperlina; avastin (bevacizumab); azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; Sodium brequinar; biririmine; busulfan; cactinomycin; calusterona; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor); chlorambucil; Corylemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; Dacarbazine; Dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; Doxorubicin hydrochloride; droloxifene; Droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomitin hydrochloride; elsamitrucin; enloplatin; enpromato; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etoprin; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; Fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosin; iproplatin; Irinotecan; Irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansina; mechlorethamine hydrochloride; Megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; sodium methotrexate; metoprine; meturedepa; mitinomide; mitocarcin; mitochromin; mitogilin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargasa; Peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; pentamethane; sodium porfimer; porphyromycin; Prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazof urina; riboprine; safingol; safingol hydrochloride; semustine; simtrazeno; sodium sparfosate; sparsoicin; Spirogermanium hydrochloride; spiromustine; spiroplatin; Streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; Teroxirone; testolactone; tiamiprine; thioguanine; thiotepa; thiazofurine; tirapazamine; Toremifene citrate; trestolone acetate; trie i ri b i na phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; Uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidin sulfate; sulfate of ving licinato; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zipiplatine; zinostatin; and zorubicin hydrochloride.

Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acilfulveno; adecipenol; adozelesina; adlesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrografol; inhibitors of angiogenesis; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen; Prostatic carcinoma; antiestrogen; antineoplaston; anti-sense oligonucleotides; afidicolin glycinate; modulators of apoptosis genes; apoptosis regulators; Apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azathirosine; Baccatin III derivatives; balanol batimastat; BCR / ABL antagonists; benzoclorins; benzoylstaurosporin; beta lactam derivatives; beta-aletine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinyl espermine; bisnafida; bistratene A; bizelesin; breflato; biririmine; budotitan; butionine sulfoximine; calcipotriol; calfostin C; camptosar (also called Campto; irinotecan) derived from camptothecin; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; inhibitor derived from cartilage; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorines; Chloroquinoxaline sulfonamide; cycaprost; cis-porphyrin; cladribine; clomiphene analogues; clotrimazole; crambescidin 816; crisnatol; cryptophycin 8; Cryptophycin A derivatives; curacin A; cyclopentantraquinones; Cycloplatam; cipemycin; cytarabine ocphosphate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidenmine B; deslorelin; dexamethasone; dexiphosphamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol; duocarmicin SA; ebselen; ecomustine; edelfosin; Edrecolomab; eflornithine; elemeno; emitefur; epirubicin; epristerida; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; Finasteride; vopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulina; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifen; idramantone; ilmofosin; ilomastat; imatinib (for example, GELLEVE®), imiquimod; immunostimulatory peptides; inhibitor of growth factor 1 receptor as insulin; interferon agonists; interferons; interleukins; iobenguan; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; Sobengazol; isohomohalicondrine B; itasetron; jasplakinolide; kahalalide F; lamelarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; Leukemia inhibitory factor; leukocyte interferon alpha; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analog; lipophilic disaccharide peptide; lipophilic platinum compounds; lisoclinamide-7; lobaplatin; lombrlcina; lometrexol; lonidamine; losoxantrone; loxoribine; lurtotecan; lutetium texaphyrin; lyophilin; UTIC peptides; Maytansine; Handstatin A; marimastat, masoprocol; maspina; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; inhibitor MY F; mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide; saporin growth factor of mitotoxin filbroblast; mitoxantrone; mofarotene; molgramostim; Erbitux (cetuximab), human chorionic gonadotropin; lipid A of monophosphoryl + myobacterial cell wall sk; mopidamol; mustard anti-cancer agent; micaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavina; nafterpina; nartograstim; nedaplatin; nemorubicin; acid neridronic; nilutamide; nisamycin; Nitric oxide modulators; nitroxide antioxidant; nitrulin; oblimersen (GENASENSE®); O6-benzylguanine; octreotide; okicenona; oligonucleotides; onapristone; ondansetron; oracine; oral cytosine inducer; ormaplatin; osaterone; oxaliplatin (e.g., Floxatin); oxaunomycin; paclitaxel; Paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxitriol; panomiphene; parabactin pazeliptina; pegaspargasa; peldesina; pentosan sodium polulose; pentostatin; pentrozole; perflubron; perfosfamide; perilyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetina A; placetina B; inhibitor plasminogen activator; platinum complex; platinum compounds; platinum-triamine complex; sodium porfimer; porphyromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; immune modulator based on protein A; inhibitor of protein kinase C; inhibitors of protein kinase C; microalgal; protein tyrosine phosphatase inhibitors; inhibitors of purine nucleoside phosphorylase; purpurins; pyrazoloacridine; polyoxyethylene conjugate of pyridoxylated hemoglobin; raf antagonists; raltitrexed; ramosetron; ras farnesyl ras transferase protein inhibitors; ras inhibitors; ras-GAP inhibitor; Demethylated reteliptine; Re 186 rhenium etidronate; rhizoxin; ribozymes; retinamide Rll; rohituquine; romurtida; roquinimex; Rubiginone B1; ruboxyl; safingol; saintopine; SarCNU; sarcofitol A; sagramostim; Sdi 1 mimetic; semustine; inhibitor derived from senescence 1; sense oligonucleotides; inhibitors of signal transduction; sizofiran; Sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; Somatomedin binding protein; sonermin; Sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stipiamide; stromelsin inhibitors; Sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista suramin; Swainsonin; talimustine; tamoxifen methylodide; tauromustine; tazarotene; tecogalan sodium; tegafur; telurapyrilio; telomerase inhibitors; temoporfin; teniposide; tetrachlorodeoxide, tetrazomine; Taliblastine; thiocoraline; thrombopoietin; mimetic thrombopoietin; timalfasin; thymopoietin receptor agonist; thymotrinam; thyroid stimulating hormone; tin ethyl etpurpurine; tirapazamine; titanocene bichloride; topsentin; toremifene; Translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; Tyrphostins; UBC inhibitors; ubenimex; growth inhibitory factor derived from urogenital sinus; Urokinase receptor antagonists; vapreotide; Variolin B; Vectibix (panitumumab) velaresol; veramina; verdinas; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; Welcovorin (leucovorin); Xeloda (capecitabine); zanoterone; zipiplatine; zilascorb; and zinostatin estimalmer. 6. 8.4. Viral Infection Treatment In another embodiment, a method of treating an individual having a viral infection, which comprises administering to said individual a therapeutically effective amount of TSNK cells, is provided herein. In certain embodiments, the individual has a deficiency of natural killer cells, for example, a deficiency of active NK cells against the individual's viral infection. In certain specific embodiments, said administration further comprises administering to the individual one or more of placental perfusate isolated, isolated placental perfusate cells, isolated natural killer cells, eg, natural killer cells of placenta, eg, derived intermediate natural killer cells of placenta, isolated combined natural killer cells and / or combinations thereof. In certain specific modalities, TSNK cells are contacted with an immunomodulatory compound, for example, an immunomodulatory compound described in 6.2.1, above, or thalidomide, prior to said administration. In certain other specific embodiments, said administration comprises administering an immunomodulatory compound, for example, an immunomodulatory compound described in section 6.2.1, above, or thalidomide, to said individual in addition to said TSNK cells, wherein said amount is an amount that is , for example, results in a detectable improvement of, reducing the progress of, or elimination of, one or more symptoms of said viral infection. In specific modalities, the viral infection is an infection by a virus of the family Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papilommaviridae, Rhabdoviridae or Togaviridae. In more specific modalities, said virus is human immunodeficiency virus (HIV), coxsackievirus, hepatitis A virus (HAV), poliovirus, Esptein-Barr virus (EBV), herpes simplex type 1 (HSV1), herpes simplex type 2 (HSV2), human cytomegalovirus (CMV), human herpesvirus type 8 (HHV8), herpes zoster virus (varicella zoster virus (VZV) or herpes virus), hepatitis B virus (HBV), hepatitis C virus (HCV) , hepatitis D virus (HDV), hepatitis E virus (HEV), influenza virus (for example, influenza A virus, influenza B virus, influenza C virus or togotovirus), measles virus, mumps virus, parainfluenza virus, papilloma virus, rabies virus or rubella virus.

In other more specific embodiments, said virus is adenovirus species A, serotype 12, 18 or 31; adenovirus species B, serotype 3, 7, 11, 14, 16, 34, 35 or 50; adenovirus species C, serotype 1, 2, 5 or 6; species D, serotype 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49 or 51; species E, serotype 4; or species F, serotype 40 or 41.

In certain other more specific modalities, the virus is Apoi virus (APOIV), Aroa virus (AROAV), bagasse virus (BAGV), Banzi virus (BANV), Bouboui virus (BOUV), Cacipacore virus (CPCV), Carey Island virus ( CIV), Cowbone Ridge virus (CRV), Dengue virus (DENV), Edge Hill virus (EHV), Gully Gadget virus (GGYV), llheus virus (ILHV), Israel turkey meningoencephalomyelitis virus (ITV), encephalitis virus Japanese (JEV), Jugra virus (JUGV), Jutiapa virus (JUTV), Kadam virus (KADV), Kedougou virus (KEDV), Kokobera virus (KOKV), Koutango virus (KOUV), Kyasanur Forest disease virus (KFDV), Langat virus (LGTV), Meaban virus (MEAV), Modoc virus (MODV), Montana myotis leukoencephalitis virus (MMLV), Murray Valley encephalitis virus (MVEV), Ntaya virus (NTAV), Omsk hemorrhagic fever virus (OHFV) ), Powassan virus (POWV), Rio Bravo virus (RBV), Royal Pharmaceutically acceptable virus (RFV), Savoy virus (SABV), St. Louis encephalitis virus (SLEV), Sal Veja virus (SVV), San Perlita virus (SPV), Saumarez Reef virus (SREV), Sepik virus (SEPV), Tembusu virus (TMUV), tick-borne encephalitis virus (TBEV), Tyuleniy virus (TYUV), Uganda S virus (UGSV), virus Usutu (USUV), Wesselsbron virus (WESSV), West Nile virus (WNV), Yaounde virus (YAOV), yellow fever virus (YFV), Yokose virus (YOKV) or Zika virus (ZIKV).

In other embodiments, TSNK cells, and optionally perfused placenta and / or perfusate cells, are administered to an individual having a viral infection as part of an antiviral therapy regimen that includes one or more other antiviral agents. Specific antiviral agents that can be administered to an individual having a viral infection include, but are not limited to: imimoimod podofilox, podophyllin, interferon alpha (IFNa), lattices, nonoxynol-8, acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine, rimantadine; ribavirin; zanamavir and oseltaumavir; protease inhibitors such as indinavir, nelfinavir, ritonavir or saquinavir; inhibitors of inverted nucleoside transcriptase such as didanosine, lamivudine, stavudine, zalcitabine or zidovudine; and non-nucleoside inverted transcriptase inhibitors such as neviparin or efavirenz. 6. 8.5. Administration The determination of the number of cells, for example, placental perfusate cells, for example, placental perfused nucleated cells, combined natural killer cells, and / or isolated natural killer cells, e.g., TSNK cells, and quantity determination of an immunomodulatory compound, for example, as an immunomodulatory compound in section 6.2.1, above, or thalidomide, can be performed independently of one another. 6. 8.5.1. Cell Administration In certain embodiments, TSNK cells are used, for example, administered to an individual, in any amount or number that results in a detectable therapeutic benefit to the individual, for example, an effective amount, wherein the individual has a viral infection, cancer or tumor cells, for example, an individual having tumor cells, a solid tumor or a blood cancer, for example, a patient with cancer. Said cells can be administered to said individual by absolute numbers of cells, for example, said individual can be administered at about, at least approximately, or at most approximately, 1 x 105, 5 x 105, 1 x 106, 5 x 106 , 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 X 109, 5 x 109, 1 x 1010, 5 x 1010 or 1 x 1011 TSNK cells. In other embodiments, the TSNK cells can be administered to said individual by relative numbers of cells, for example, said individual can be administered at about, at least approximately, or at most approximately, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010 or 1 x 1011 TSNK cells per kilogram of the individual. TSNK cells can be administered to said individual according to an approximate ratio between a number of TSNK cells, and optionally placental perfusate cells and / or natural killer cells other than TSNK cells, and a number of tumor cells in said individual (eg, example, an estimated number). For example, TSNK cells can be administered to said individual in a ratio of about, at least about or at most about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1, 65: 1, 70: 1, 75: 1, 80: 1, 85: 1, 90: 1, 95: 1 or 100: 1 to the number of tumor cells in the individual. The number of cells Tumor in said individual can be estimated, for example, by counting the number of tumor cells in a tissue sample from the individual, for example, blood sample, biopsy, or the like. In specific modalities, for example, for solid tumors, said counting is performed in combination with images of the tumor or tumors to obtain an approximate tumor volume. In a specific embodiment, an immunomodulatory compound or talimodomide, for example, an effective amount of an immunomodulatory compound or thalidomide, are administered to the individual in addition to the TSNK cells, optionally placental perfusate cells and / or natural killer cells other than the TSNK cells.

In certain embodiments, the method of suppressing the proliferation of tumor cells, for example, in an individual; treatment of an individual having a deficiency in the natural killer cells of the individual; or treatment of an individual having tumor cells, a blood cancer or a solid tumor, comprises contacting the tumor cells, or administering to said individual, a combination of TSNK cells and one or more perfusate of placenta and / or perfusate cells of placenta. In specific embodiments, the method comprises additionally contacting the tumor cells, or administering to the individual, an immunomodulatory compound or thalidomide.

In a specific embodiment, for example, treatment of an individual having a deficiency in the natural killer cells of the individual (eg, a deficiency in the number of NK cells or in the reactivity of NK cells to a cancer, tumor or cell virally infected); or treatment of an individual having a cancer or a viral infection, or suppression of tumor cell proliferation, comprising contacting said tumor cells, or administering to said individual, TSNK cells supplemented with placental perfusate cells isolated or perfused from placenta. In specific modalities, approximately, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more TSNK cells per milliliter, or, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010 or 1 x 1011 or more TSNK cells, are supplemented with approximately, or at least approximately, 1 x 105, 5 x 105, 1 x 106 , 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108 or more isolated placental perfusate cells per milliliter, or, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010 or 1 x 1011 0 more isolated placental perfusate cells. In other more specific modalities, approximately 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108 or more TSNK cells per milliliter, or, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 X 108, 5 x 108, 1 x 109, 5 x 109, 1? 1010, 5? 1010 or 1 x 1011 or more TSNK cells are supplemented with approximately, or at least approximately, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ml_ of perfusate, or approximately 1 unit of perfusate.

In another specific embodiment, the treatment of an individual having a deficiency in the natural killer cells of the individual; treatment of an individual who has cancer; treatment of an individual having a viral infection; or suppression of tumor cell proliferation, comprises contacting the tumor cells, or administering to the individual, TSNK cells, wherein said cells are supplemented with adherent placental cells, for example, placental perfused stem cells or multipotent cells, for example, Placental cells adherent to plastic culture CD34 \ CD10 \ CD105 +, CD200 +. In specific embodiments, TSNK cells are supplemented with approximately 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 1 O8 or more placental stem cells adherent per milliliter, or, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010 or 1 x 1011 or more adherent placental cells, e.g., adherent placental stem cells or multipotent cells.

In another specific embodiment, the treatment of an individual having a deficiency in the natural killer cells of the individual; treatment of an individual who has cancer; treatment of an individual who has a viral infection; or suppression of tumor cell proliferation, is performed using an immunomodulatory compound or thalidomide in combination with TSNK cells, in wherein said cells are supplemented with conditioned medium, for example, conditioned medium by placental cells adherent to tissue culture plastic CD34", CD10 +, CD105 +, CD200 +, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 , 0.7, 0.8, 0.9, 1. 2, 3, 4, 5, 6, 7, 8, 9, 10 mL of culture medium conditioned by stem cell per perfusate unit, or by 104, 105, 106, 107, 108, 109, 1010 or 1011 TSNK cells In certain embodiments, the tissue-adherent plastic placenta cells are the multipotent adherent placental cells described in US Patent No. 7,468,276 and US Patent Application Publication No. 2007/0275362, the descriptions of which are incorporated herein by reference in their entireties In another specific embodiment, the method further comprises contacting the tumor cells, or administering to the individual, an immunomodulatory compound or thalidomide.

In another specific embodiment, the treatment of an individual having a deficiency in the natural killer cells of the individual; treatment of an individual who has cancer; treatment of an individual who has a viral infection; or suppression of tumor cell proliferation, wherein said TSNK cells are supplemented with placental perfusate cells, the perfusate cells are contacted with interleukin-2 (I L -2) for a period of time before said contact. In certain modalities, said period of time is approximately, at least, or at least much 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 , 42, 44, 46 or 48 hours before said contact.

TSNK cells, and optionally perfused or perfusate cells, can be administered once to an individual having a viral infection, an individual having cancer, or an individual having tumor cells, during a course of anti-cancer therapy.; or they can be administered multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22 or 23 hours, or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 24, 36 or more weeks during therapy. In embodiments, wherein cells and an immunomodulatory compound or thalidomide are used, the immunomodulatory compound or thalidomide, and cells or perfusate, can be administered to the individual together, for example, in the same formulation; separately, for example, in separate formulations, at about the same time; or they can be administered separately, for example, at different dosing schedules or at different times of the day. Similarly, in embodiments where cells and an antiviral compound or anti-cancer compound are used, the antiviral compound or anti-cancer compound, and cells or perfusate, can be administered to the individual together, for example, in the same formulation; separately, for example, in separate formulations, at about the same time; or they can be administered separately, for example, in different dosing schedules or at different times of the day. TSNK cells, and perfused or Perfusate cells can be administered regardless of whether the TSNK, perfused or perfused cells have been administered to the individual in the past. 7. EXAMPLES 7. 1. Example 1: Recovery of Hematopoetic Stem Cells from Human Placenta Perfusate and Umbilical Cord Blood Typically perfused human placenta (HPP) and umbilical cord blood (UCB) were purified using Ficoll and ammonium chloride to obtain total nucleated cells (TNCs). TNCs were then used in a positive selection procedure to isolate CD34 + cells using anti-CD34 and RoboSep spheres according to the manufacturer's protocol (StemCell Technologies, Inc.). In this experiment, CD34 + cells with more than 90% purity were isolated. Alternatively, the EasySep® Human Progenitor Cell Enrichment Kit (StemCell Technologies, Inc.) was used in a negative selection procedure to bypass cells committed to lineage by using Human Progenitor Cell Enrichment Cocktail with monoclonal antibodies to the following human cell surface antigens: CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A. Using the negative selection, 90% of CD34 + cells were recovered from the raw material. The cellular composition of the recovered HSCs is summarized in Table 1.

Table 1. Cell composition of hematopoietic stem cells (HSCs) enriched. The standard deviation was calculated for the population medium of 3 donors. 7. 2. Example 2: Cell-free Expansion and Differentiation Hematopoietic Stem Cell Feeder in Natural Killer Cells CD34 + cells were cultured in the following medium formulations for up to 48 days, and aliquots of cells were taken for evaluation of cell count, cell viability, characterization of differentiation and functional evaluation of natural killer cells.

NK medium: GBGM (Growth medium based on Glycostem, Glycostem Cat # CCT-SCB500, transparent cellular technology) supplemented with pen / strep (Cat # 15140, Gibco), 20 ng / mL SCF (Cat # 255-SC, R &D Systems), 10 ng / mL ligand Flt-3 (Cat # 308-FK, R &D Systems), 20 ng / mL TPO (Cat # 288-TP, R &D Systems), 20 ng / mL IL -7 (Cat # 207-IL, R &D Systems), 200 lU / mL IL-2 (Cat # 202-IL, R &D Systems) and 10 ng / mL IL-15 (Cat # 247-IL, R &; D Systems).

NK2 medium: DMEM (Cat # MT-10-013-CV, Fisher): Ham's F12 (Cat # BW12-615F, Fisher) as 1: 2 supplemented with 2 mM L-Glutamine (Cat # 25030, Invitrogen), 1% pen / strep, 20% human serum AB (Cat # 100-512, Gemcell), 5 ng / mL sodium selenite (Cat # S9133, Sigma), 50 μ? Ethanolamine (Cat # E0135, Sigma), 25 μ? β-mercaptoethanol (Cat # 21985, Invitrogen), 20 ng / mL ascorbic acid (Cat # 47863, Sigma), 5 ng / mL IL-3 (Cat # 203-IL, R &D Systems), 20 ng / mL SCF , 10 ng / mL ligand Flt-3, 20 ng / mL IL-7 and 10 ng / mL IL-15.

NK3 medium: X-vivo 20 (Cat # BW04-448Q, Fisher) supplemented with pen / strep, 10% human serum AB (Cat # 100-512, Gemcell) and 500 lU / mL IL-2.

NK2A medium: GBGM supplemented with 10% human serum AB, 1% pen / strep, 20 ng / mL SCF, 10 ng / mL ligand Flt-3, 20 ng / mL TPO, 20 ng / mL IL-7, 200 lU / mL IL-2, 10 ng / mL IL-15 and 1.5 lU / mL heparin (Cat # H3149, Sigma).

NK2B1 medium: DMEM: Ham's F12 as 1: 2 supplemented with 2 mM L-glutamine, 1% pen / strep, 20% human AB serum, 5 ng / mL sodium selenite, 50 μ? Ethanolamine, 25 μ? β-mercaptoethanol, 20 μg / mL ascorbic acid, 5 ng / mL IL-3, 20 ng / mL SCF, 10 ng / mL ligand Flt-3, 20 ng / mL IL-7 and 10 ng / mL IL-15.

NK2B2 medium: DMEM: Ham's F12 as 1: 2 supplemented with 2 (IIM L-glutamine, 1% pen / strep, 20% human AB serum, 5 ng / mL sodium selenite, 50 μ? Ethanolamine, 25 μ? β-mercaptoethanol, 20 μg / mL ascorbic acid, 200 μU / mL IL-2, 20 ng / mL SCF, 10 ng / mL ligand Flt-3, 20 ng / mL IL-7 and 10 ng / mL IL-15.

NK2C medium: RPMI 1640 (Cat # 22400105, Invitrogen) supplemented with 10% FBS (Cat # SH30070.03, Hyclone), 2 mM L-glutamine, 1% pen / strep, 50 ng / mL SCF, 50 ng / mL Ligand Flt-3, 100 IU / mL IL-2, 20 ng / mL IL-7 and 20 ng / mL IL-15.

NK2D medium: serum free medium (StemSpan, Cat # 09650, Stem Cell Technologies, Vancouver, Canada) supplemented with 1 μ? Synthetic glucocorticoid dexamethasone (Dex, Cat # D4902, Sigma, St. Louis, MO), 40 ng / mL insulin-like growth factor 1 (IGF-1, Cat # 291-G1-250, R &D Systems, Minneapolis, MN ), 100 ng / mL SCF, 40 ng / mL lipids (mixture of lipid-rich co-cholesterol, Cat # C7305-1G, Sigma, St Louis, MO), 5 ng / mL IL-3, 200 lU / mL IL-2 , 20 ng / mL IL-7 and 20 ng / mL IL-15.

The harvested cells were washed at different time points twice with TPM11640 (phenol-free) and 5% FBS, labeled with fluorescence-conjugated antibodies (Tables 2 and 3) for 15 minutes at 4 ° C, and analyzed by flow cytometry. (FACSCanto, BD) and flow cytometry software Jo (Tree Star).

Table 2. Antibodies used for cell tag Table 3. NK surface receptor flow cytometric characterization panel Cytotoxicity assay using PKH26 / TO-PRO-3 tag. Target tumor cells were labeled with PKH26 (Cat # PKH26GL, Sigma-Aldrich), a dye that inserts into cellular plasma membrane via its lipophilic lytic residue, then placed in 96-well U-bottom tissue culture plates. incubated with expanded NK cells at various effector-objective relationships (E: T) at 200μ? RPM! 1640 supplemented with 10% FBS. Cultures were incubated for 4 hours at 37 ° C in 5% C02. After incubation, cells were harvested and TO-PRO-3 (Invitrogen Cat # T3605), a DNA stain impermeable to membrane, was added to cultures (1 μm final concentration) followed by FACS analysis. Cytotoxicity was expressed as the percentage of dead cells (PKH26 + TO-PRO-3 +) within the total PKH26 + target tumor cells.

Optimization of expansion and differentiation of CD34 * cells in NK cells. Among the media tested formulations, media NK1, NK2, and NK3, NK1 showed an expansion of 500 folds on day 21 (D21). Neither the NK2 nor NK3 medium maintained cell proliferation or differentiation. More media optimization was performed for NK1 medium and subsequent media were called NK2A, NK2B, NK2C and NK2D,. CD34 + cells cultured with NK2A medium showed an expansion of 10"5-fold on day 55 (D55). Based on the results of fold expansion, differentiation and cytotoxicity of NK1, 2 and 3 medium, the second batch of Formulations of NK2A, NK2B, NK2C and NK2D media were made and in D55, a 105-fold expansion was achieved (as shown in Figure 1.) The NK2B medium showed an expansion of approximately 3 x 10-fold. in NK2C medium resulted in expansion of 3 x 102-fold in 21 days, followed by decreased cell viability.NK2D medium did not maintain cells throughout the duration of the experiment.

On day 48 (D48), about 90% of the NK cells in NK2A medium were CD56 + CD3 ~. Within the population CD56 + CD3", more than 98% of cells were CD56 + CD16" (as shown in Figure 2), while 58% expressed the activating receptor NKG2D, 68% were NKp46 + and 17% were CD226 + ( as shown in tables 4A and 4B).

Table 4A, 4B. Phenotypic characterization of expanded NK cells in D48 Additionally, 97.8% of cells cultured in NK2A medium and 93.1% of cells cultured in NK2B medium were CD56 + CD16"on day 21 of culture. 7. 3 Example 3: Culture of NK Cells in CNK Medium Power Expansion and Cytotoxicity of NK Cells On day 27 (D27), CD34 + cells cultured in the NK2A medium were further cultured in one of the following media: • Two-stage medium, comprising CNK medium and Maintenance Medium. CNK medium is IMDM (Invitrogen) supplemented with 10% FCS (Hyclone), 200 lU / mL IL-2 (R &D Systems), 35 Mg / mL transferin (Sigma-Aldrich), 5 μg / mL insulin (Sigma- Aldrich), 2 x 10'5 M ethanolamine (Sigma-Aldrich), 1 μ9 /? Oleic acid (Sigma-Aldrich), 1 μg / mL linoleic acid (Sigma-Aldrich), 0.2 μg / mL palmitic acid ( Sigma-Aldrich), 2.5 μ9 / β-BSA (Sigma-Aldrich) and 0.1 μg / mL phytohaemagglutinin (PHA-P, Sigma-Aldrich). CD56 + CD3"NK cells cultured in NK2A medium were suspended at 2.5 x 10 5 live cells / mL in CNK medium in 24-well plates treated with cell culture or T-bottles. Allogeneic PBMC treated with mitomycin C and K562 cells (line chronic myelogenous leukemia cell) were added to the medium of CNK as feeder cells, at a final concentration of 1 x 106 per mL. NK cells were cultured for 5-6 days at 37 ° C in 5% C02. After 5-6 days and then every 3-4 days a volume equal to maintenance medium (IMDM with 10% FCS, 2% AB serum) human, antibiotics, L-glutamine and 400 units of IL-2 per ml_) was added to! culture.

• NK2A medium (PDAC) with placental stem cells adherent to tissue culture plastic CD34", CD10 +, CD105 +, CD200 + treated with mitomycin C as feeder cells; • NK2A medium (MSC) with mesenchymal stem cell (MSC) treated with mitomycin C as feeder cells; or · Feeding free NK2A medium (FF) as the control.

The two-stage medium enhanced the fold expansion of CD34 + cells compared to NK2A (FF), NK2A (PDAC) and NK2A (MSC), in particular between day 27 (D27) and day 48 (D48). See figure 3.

By day 35, the proportion of CD34 + cells had already decreased to approximately 4% while the proportion of CD56 + CD3"had increased to approximately 80% in the two-stage medium." Day 45 (D45), the cells cultured in Two-stage media showed higher cytotoxicity compared to NK cells treated in NK2A (FF), NK2A (PDAC) and NK2A (MSC) (as shown in Figure 3) .Phenotypic characterization on day 41 (as shown) in Figure 5) showed increased expression of NKp46 and CD226 in the cells, indicating a possible explanation for the improvement of cytotoxicity.In D41, the proportion of CD226 + cells increased from 0.9% ± 0.8% in NK2A medium to 13% ± 4% in the middle of two stages, the proportion of NKp46 + cells increased from 55.4 ± 8.7% in NK2A medium to 80% ± 7.85% in two-stage medium In D48 the proportion of CD226 + cells increased from 17.3 ± 14.3% in mean of NK2A to 52.3% ± 11.64% in the middle of two stages; NKp46 + cells increased from 67.9% ± 5.4% in NK2A medium to 86% ± 4% in two stages. There was no significant difference in NKG2D expression between the conditions tested. The changes in expression of CD226 and NKp46 are shown in Table 5, below.

Table 5. Expression of CD226 and NKp46 in NK cells cultured in NK2A (FF) and two-stage medium on day 41 (D41) and day 48 (D48). The standard deviation was calculated for 3 donors. 7. 4 Example 4: Comparison of Natural Killer Cells Cultivated in Two Step Medium and Natural Killer Cells Derived from Embryonic Stem Cells (ESCs) NK cells in the two-stage medium were compared with NK cells derived from embryonic stem cells (ESCs), which were produced by the method of Woll et al., Blood 113 (4): 6094-6101 (2009). Specifically, a difference in expression levels of CD94 and CD117 was observed during the culture process of both cell types. Figure 6 shows that CD117 expression was high in two-step NK cells, or "+", from Day 7 (D7) to Day 35 (D35), while CD94 expression increased gradually. At day 35 (D35), approximately 44% of the CD56 + CD3"(two steps) 'cells were CD94 + CD117 \ 37.6% of CD56 + CD3 cells were CD94" CD117 + and 14.7% of CD56 + CD3 cells "They were CD94 + CD117 cells". As such, the NK cells produced by the two-step method are distinguishable from the NK cells derived from ESCs, 78% of which remained CD117baio / "from day 14 to day 35 of the culture.This difference in CD117 expression is already useful. that NK cells CD117 + are cytotoxic towards tumor cell lines of various tissues, as described in Example 6, below.

These results suggest that the progress of differentiation of TSNK cells is different from NK cells derived from ESC, and that TSNK cells are distinguishable from NK cells derived from ESC. 7. 5 Example 5: PDACs Improve Fold Expansion of Natural Killer Cells Cultivated in NK2A Medium To evaluate the effect of placental stem cells adherent to tissue culture plastic CD10 +, CD34", CD105 +, CD200 + (referred in this example as PDACs) in differentiation of hematopoietic stem cell (HSC) to natural killer cells, HSCs were stimulated with PDACs treated with mitomycin C or mesenchymal stem cells derived from bone marrow (MSC) at a ratio of 10: 1 PDACs / MSC: HSC on day 0 and day 21, while a feeder-free culture was used as the control NK2A medium was used as a culture medium PDACs were found to improve the expansion of fold of cultured NK cells compared to medium alone, however, no significant difference in cytotoxicity was found between cells grown with or without the feeder layer.The cells treated with MSC showed the highest fold expansion, but the lowest cytotoxicity , as shown in figure 7. 7. 6 Example 6: Cytotoxic Activity of Expanded NK Cells Using Two Stage Media This example demonstrates that the NK cells produced from CD34 + cells expanded and differentiated using the two-step process described above are cytotoxic to tumor cell lines.

Dehydrogenase release test of lactate (LDH). The LDH release assay was performed using CYTOTOX 96® non-radioactive cytotoxicity equipment (Promega, Cat # G1780). In this assay, cultured NK cells derived from donor-compatible human placenta perfusate (HPP) and cord blood units (Combos units) were used as effector cells, while certain tumor cell line cells were used as target cells. From three units in this study, the percentage of HPP cells was 56.6 ± 28.3%. Effector cells and target cells were placed in 96-well U-bottom tissue culture plates and incubated at various effector-target (E: T) ratios at 100 μ? RPMI 1640 without red phenol (Invitrogen, Cat # 11835-030) supplemented with 2% human serum AB (Gemini, Cat # 100-512). Cultures were incubated for 4 hours at 37 ° C in 5% C02. After incubation, 50 μ? of supernatant was transferred to enzyme assay plate, LDH activity was detected as provided by the manufacturer, and absorption was measured at 490 nm in an ELISA reader (Synergy HT, Biotek). The cytotoxicity was calculated according to the following equation:% Cytotoxicity = (Sample = Spontaneous Efectora - Spontaneous Diana) / (Maximum Diana - Spontaneous Diana) * 100, where "Spontaneous Efectora" is a control for the spontaneous release of LDH from effector cells; "Spontaneous Diana" is a control for the spontaneous release of LDH from target cells; and "Diana Maximum" is a control for maximum LDH release when essentially 100% of the cells are lysed.

Isolation of CD34 + Human Placenta Perfused Cell (HPP) and CD34 + Umbilical Cord Blood Cell (UCB). HPP and UCB cells were purified using Ficoll or ammonium chloride to obtain total nucleated cells (TNCs). TNCs were then used in a positive selection procedure to isolate CD34 + cells using anti-CD34 and RoboSep spheres following the protocol provided by the manufacturer (StemCell Technologies, Inc.). In this experiment, CD34 + cells with more than 90% purity were isolated.

Tumor cell susceptibility study to two-stage NK cells cultured. Tumor cell lines (table 1), including human breast cancer (HCC2218), human colorectal adenocarcinoma (HT-29), human chronic myelogenous leukemia (CML), human acute myeloid leukemia (AML), human glioblastoma (LN-18 and U-118MG), human multiple myeloma (U266), human histiocytic lymphoma (U937), and human retinoblastoma (WERI-RB-1) were co-cultured in two-stage NK cells. NK cells cultured in two stages included those cultured in NK2A medium for 21 days, then cultured in CNK medium for 21 days, and those cultivated in NK2A medium for 28 days and then CNK medium for 14 days. The NK cell cytotoxicity was measured by the lactate dehydrogenase (LDH) release assay after 4 hours co-culture. At an effector to target ratio (E: T) of 10: 1, the latter usually showed greater cytotoxicity than the first (Table 6). Of the tumor cell lines, LN-18 was the most susceptible to killing mediated with NK, followed by K562, U937, WERI-RB-1, U-118MG, HT-29, HCC2218, KG-1 and U266.

Therefore, TSNK cells showed significant cytotoxicity towards several cancer cell lines. Furthermore, it seems that the NK cytotoxicity can be improved by prolonging the culture period in NK2A medium from 21 days to 28 days.

Table 6. Cytotoxicity of cultured TSNK cells focusing on tumor cell lines MicroRNA profile of Human Placenta Perfused CD34 + Cells (HPP) and CD34 + Umbilical Cord Blood Cells (UCB). Purified donor-compatible HPP and CD34 + cells were subjected to microRNA (miRNA) preparation using a miRNA IRVANA ™ isolation kit (Ambio, Cat # 1560). CD34 + cells (0.5 X 10 6 cells) were disrupted in a denaturing lysis regulator. The samples were then subjected to acid-phenol + chloroform extraction to isolate highly enriched RNA for small RNA species. 100% ethanol was added to take the samples to 25% ethanol. When this mixture of lysate / ethanol passed through a glass fiber filter, large RNA species were immobilized, and the small RNA species were collected in the filtrate. The ethanol concentration of the filtrate was then increased to 55%, and the mixture passed through a second glass fiber filter where the small RNAs are immobilized. This RNA was washed few times, and eluted in a low resistance ionic solution. The concentration and purity of the recovered small RNA was determined by measuring its absorbance at 260 and 280 nm. It was discovered that miRNAs were unique for CD34 + HPP cells in all tested donors (n = 3) including hsa-miR-380, hsa-miR-512, hsa-miR-517, hsa-mR-518c, hsa-miR-519b, and hsa-miR-520a. 7. 7 Example 7: Isolation of CD34 * cells from UCB and HPP pooled This example demonstrates the isolation of CD34 + cells from umbilical cord blood (UCB) and perfused human placenta (PPH) grouped (Combo). To evaluate the grouping relationship of UCB: HPP, side-by-side comparisons were made from 3 different grouping ratios as follows: (1) full grouping: 1x UCB (full volume) + 1x HPP (full volume); (2) partial grouping of HPP (33%): 1x UCB (full volume) + 0.33x HPP (1/3 volume HPP); and (3) partial grouping of HPP (10%): 1x UCB (full volume) + 0.10x HPP (1/10 volume HPP). A total of N = 3 experimental replicas was executed. Initial TNC and volumes were recorded. The pooled samples were then purified for CD34 + cells and CD34 + purity was determined post-thawing. The optimal grouping ratio was then determined graphically of the purity CD34 + post-thawing vs. Volumetric fractions or cell count (TNC) (as combo%) graphs. As shown in Figure 8, the endpoint CD34 + purity correlates well with the volume content of HPP, but not so much with the HPP TNC content. Overall, UCB 85% v / v, HPP 15% v / v was found to be the optimal grouping ratio to obtain CD34 + cells with purity of 80% and more. 7. 8 Example 8: Comparison of NK Cell Culture Using medium based on GBGM® This example demonstrates the comparison of NK cell culture processes using two GBGM-based media (the three-step process and the two-step process). The two processes are summarized in Table 10. Both processes used GBGM as the basal medium for the differentiation of NK cells and CD34 + cells from placenta in origin.

Table 7. Summary of the processes of three stages and two stages The experimental parameters are outlined below: Lots of donors: (1) CD34 + cells from fresh UCB: N = 6 (2) Cool Combo CD34 + cells: N = 2 (3) Cryoconserved "Combo" CD34 +: N = 8 Scale: Multi-bowl dishes to T-25, up to multiple bottles T-75, mL in culture volume Process Methods: (1) Two stage process (2) Three stage process Use of feeders: (1) Without feeders (2) With K562 & Inactivated PBMC, added to culture on day 21 Seedbed density: 20000 - 50000 cells / mL CD34 + cells from fresh UCB or fresh combo were generated and cryopreserved with methods described in Examples 7, 10 and 11. Cultures were maintained in an incubator at 37 ° C, > 90% humidity and 5% C02. Cell growth was monitored (cell count) and media exchanges were made twice a week to maintain cell concentrations within the range of 5 x 104 - 1 x 106 per ml_. The differentiation was monitored by phenotypic analysis on day 21 and 35. When feeders were used, on day 21 of NK culture, fresh 3-day cultivated K562 and post-thawing allo-PBMC with mitomycin-C (16 μg) were inactivated. mL> 2 hours, 37 ° C) and were added to the two stage process conditions at 1 x 106 per mL. On day 35, after performing final cell counts, a cytotoxicity assay based on flow cytometry using K562 cells labeled with PKH and freshly cultured (ratio E: T 10: 1, 4 hr, 37 ° C) was performed to evaluate NK functionality.

Results The mean of the TNC expansion fold for CD34 + cells derived from fresh UCB using the two processes was comparable at day 35. The two-step process appeared to produce more TNC expansion fold than the three-step process for CD34 + cells derived from cool combo The TNC global expansion folds were comparable for CD34 + cells derived from post-thawing combo using both processes, but were significantly lower than those derived from fresh UCB or fresh combo. In all, both two-stage and three-stage processes produced similar cellular performance at the end of the culture.

On day 21, there were no notable phenotype differences in cells originating from UCB or Combo. The two-step process resulted in a higher percentage of CD56 + CD3 NK cells (average 14.7%) than the three-stage process (average 6.1%) .The degree of differentiation (e.g., CD56 + CD3 ~ level) be dependent on donor The percentage of both populations CD3 + CD56"(T cells) and CD3 + CD56 + (NKT type cells) were minimal in all cases.

On day 35, it was discovered that both processes are effective in differentiating NK cells, as evidenced by the CD56 + CD3 purity levels of "high endpoint (87.2% and 90.1% for the two-stage and three-stage processes respectively The addition of feeders in the two-stage process appeared to improve the phenotypic purity of NK (75.3% without feeders, 87.2% with feeders), although no observable benefit of feeders was found on NK purity (90.1% without feeders; % with feeders) with the three-stage process.

Cultured NK cells maintained their production of CD16 'phenotype. The two-step process appeared to produce slightly more CD56 + CD3 + cells in the absence of feeders (average 11.2%) than the other conditions (< 2%). The presence of PBMC and K562 feeders in both processes significantly up-regulated / activated certain functional NK markers (NKp46, DNAM-1, CD94) in the NK cell population. Overall, the purity expression profiles of NK and functional marker were found to be compatible between the two processes, when the feeder conditions were identical.

The functionality of cultured NK cells, as determined by the in vitro 4-hour K562 cytotoxicity assay, was found to be comparable between the two processes when the feeder conditions were identical. The NK cells activated with feeder were highly effective in killer K562 cells in vitro, with average specific lysis of 93.2% and 93.6% specific lysis for the two-stage and three-stage processes, respectively.

In summary, the two-step and three-step processes were found to produce comparable growth, phenotype (purity and activation markers), and in vitro functionality for NK cells when the same feeder condition was used. The two-stage process offers the ease and convenience of growing NK cells compared to the three-stage process. 7. 9. Example 9: Comparison of NK Cell Culture Using Various Basal Media This study seeks to evaluate the differentiation and expansion of NK cells derived from CD34 + using different basal media.

The experimental conditions are summarized in Table 11. Cells were cultured as described in Example 11. All experiments were placed on the scale of multi-well dishes / T-flasks and were maintained at 37 ° C, > 90% humidity, and 5% C02 incubator. Cell growth was monitored (cell count) and media exchanges were made twice a week to maintain cell concentrations within the range of 5x104 - 1x106 per mL. The differentiation was monitored by phenotypic analysis on day 21 and day 35. On day 21, K562 cultured in 3 days fresh and post-thawing allo-PBMC were inactivated and added to growing NK cell culture at 1x106 per mL. The NK cells were normalized to 0.5x106 per mL. On day 35, after final cell counts were performed, a cytotoxicity assay based on flow cytometry using K562 cells labeled with PKH and freshly cultured (ratio E: T 10: 1, 4 hr, 37 ° C) was carried out to evaluate the functionality of NK.

Table 8. Summary of experimental conditions for evaluation of several basal media Growth Performance Stemspan H3000 and OpTimizer showed comparable growth performance (TNC expansion fold) to GBGM at day 35. Cellgro, X-VIVO 15, AIM-V, X-VIVO 10, DMEM: F12, DMEM: F12 the 5 mM OAC showed growth performance lower than GBGM.

Phenotypic analysis On day 35 (endpoint), GBGM produced approximately 80% purity of CD56 + CD3 cells. "OpTimizer and Stemspan H3000 produced approximately 50% CD56 + CD3 + DMEM purity: F12 produced approximately 35% CD56 + CD3 purity AIM-V, X-Vivo 10, X-VIVO 15 and half Cellgro produced approximately <30% purity of CD56 + CD3 'cells. The addition of OAC to DMEM: F12 basal medium during culture was found to greatly enhance the end-point NK purity, the percentage of CD56 + CD3 'on day 35 of the culture increased from 35% to 72%.

Cito toxicity / Functionality The addition of 5 mM OAC to DMEM: F12 basal medium during culture was found to greatly enhance the activation status and in vitro functionality of NK cells. The addition of OAC also significantly increased the level of NK activation markers NKp46, NKG2D, DNAM-1 and CD94. The in vitro functionality (K562 cytotoxicity) of day 35 NK cells also increased substantially, from 21.4% to 97.1%.

Overall, the NK cell properties improved significantly from the addition of 5 mM OAC. 7. 10. Example 10: Storage and Cryoconservation of NK Cells This example demonstrates the methods of storing and cryopreservating NK cells. The CD34 + hematopoietic stem cells isolated from human placental perfusate (HPP) and umbilical cord blood cells were expanded and differentiated into NK cells using the protocols described in the previous examples. The cells were cryopreserved immediately after being isolated from HPP and umbilical cord blood (on day 0) or during the first growth phase of NK cells (on day 9, day 14, day 21 or day 35 post isolation).

The cells were cryopreserved in the following cryopreservation formulations: Formulation 1 - dextran cryo medium: 5% DMSO (Sigma Aldrich, D2650), 55% dextran (10% w / v in normal saline) (10% LMD in 0.9% sodium chloride injection, Hospira), 40% HAS (Octapharma); Formulation 2 - half of trehalose cryo: 5% DMSO, 55% trehalose (10% w / v in normal saline), 40% HSA; Formulation 3 - CryoStor® CS2 (BioLife Solutions); Formulation 4 - CryoStor® CS5 (BioLife Solutions); Formulation 5 - CryoStor® CS10 (BioLife Solutions); Formulation 6 - free serum freezing medium (Sigma-Aldrich, Cat # 6295); Formulation 7 - Glycerol freezing medium (Sigma-Aldrich, Cat # C6039); or Formulation 8 - DMSO and free serum freezing medium (Sigma-Aldrich, CAT # 2639).

Cells harvested at different time points were washed several times with culture medium or saline. The cells were then centrifuged to obtain cell pellets. The supernatants were removed, and the cell pellets were suspended with cryopreservation medium at approximately 1 x 106-1.5 x 10 7 or more cells per milliliter. The cell suspension was aliquoted to 1mL or 2mL septum flasks and incubated at 2-8 ° C for approximately 10 minutes. Subsequently, the cells were frozen in a control speed freezer (Thermo) to 0. 5 ° C / min. Frozen flasks were transferred to a cryogenic freezer for vapor storage of liquid nitrogen. The cryopreserved NK cells were thawed rapidly in a 37 ° C water bath with careful turbulence of the samples until all the visible ice melted. The cell samples can be diluted with pre-heated culture medium. 7. 11. Example 11: Storage and Cryoconservation of NK Cells This example demonstrates another method of storing and cryopreservating NK cells. The CD34 + hematopoietic stem cells isolated from human placental perfusate (HPP) and umbilical cord blood cells were expanded and differentiated into NK cells using the protocols described above. Cells were cryopreserved immediately after being isolated from HPP and umbilical cord blood (on day 0) or during the first growth phase of NK cells (on day 9, day 14, day 21 or day 35 after isolation).

A cell suspension solution was prepared by combining Dextran-40 and HSA in the ratio of 60% Dextran-40 v / v, 40% HSA (from a 25% solution) v / v).

A solution of 2x thawing was prepared with 50% Dextran-40 v / v, 40% HSA (25% solution) v / v, 10% DIVISO v / v. DMSO was first added slowly to Dextran-40 and mixed well.

Subsequently, 25% HSA solution was added to the solution slowly with mixing. The resulting solution was mixed well and brought to room temperature before use.

Cryopreservation procedure The cell number was estimated and normalized as a cell suspension at 15x106 in cell suspension solution. The volume of the cell suspension was determined and an equal volume of freshly prepared 2x freezing solution was recorded and distributed to a number of bottles. MycoAlert test was performed in culture supernatants saved to detect mycoplasma contamination. A post-thaw test was performed in a retention bottle to determine post-thaw viability, cell recovery and cell characterization. 7. 12. Example 12: Analysis of cryopreserved / thawed NK cells Feasibility test. The NK cells cryopreserved in various formulations as in example 10 or 11 were thawed. Cells were frozen at the density of 2 x 106-3 x 107 cells / mL. The thawed NK cells were evaluated for cell viability using the Countess® automated cell counter (Invitrogen) on day 0, day 3 and day 18 after thawing compared to fresh cells or pre-frozen cells. Briefly, 10 μ? of cell samples were mixed with 10 μ? of blue triptan. The cell mixtures were pipetted into the Countess® camera slide. The slide was inserted into the instrument and the cells were counted. Post-freeze-thaw cells showed cell viability approximately 80% at > 90% viability, depending on different formulations of cryopreservation.

Apoptosis trial. The thawed NK cells were also evaluated for apoptosis using BD AnnV / PI Apoptosis assay kit on day 0, day 3 and day 18 after thawing. Briefly, cells were washed twice with cold 1x PBS and re-suspended in 1x binding buffer (BD Annexin V / PI kit Apoptosis part number 556547 Bonding composition regulator part number 51-66121 E 0.1M Hepes / NaOH (pH 7.4), 1.4M NaCl, 25m CaCl2 For 2x diluted 1 part 10x regulator to 9 parts of distilled water). 100μ? of cell suspension containing approximately 100,000 cells was transferred in a FACS tube. 100μ? of 1x union regulator, 5μ? of Ann V-FITC, and 5μ? of PI-PE was added to the tube. The tube was then mixed in a vortex carefully and incubated in the dark for 15 minutes. Subsequently, 400 μ? of 1x binding regulator was added. The samples were analyzed in 1 hour. The controls used to establish quadrants and grids were non-stained cells, cells stained with Ann V-FITC only and not Pl, cells stained with Pl only and not AnnV. The apoptotic cells were quantified as% of the population of lattice cases as "cells" in the size dispersion chart (FSC vs SSC). Post-freeze-thaw cells showed approximately 5-25% of dead / late apoptotic cells and 10-25% of early apoptotic cells, depending on different cryopreservation formulations. Overall, formulation 1 (5% DMSO, 55% dextran (10% w / v in normal saline), 40% HSA), formulation 2 (5% DMSO, 55% trehalose (10% w / v in saline) normal), 40% HSA), formulation 4 - CryoStor® CS5 (BioLife Solutions), and formulation 5 (CryoStor® CS10 (BioLife Solutions) showed greater cell viability and fewer apoptotic cells compared to other formulations. 7. 13. Example 13: Evaluation of Cryopreserved Cellular Banking Process This example demonstrates the evaluation of In-Process Cryoconserved Cellular Banking. Cell culture was started with UCB CD34 + cells using the method as described by Spanholtz et al., PLoS One 5 (2): e9221 (2010) using HS-AB or FBS as the source of serum. The cell concentration was determined and adjusted, and the medium was replenished as necessary. On day 7, 9, 10 or 14, approximately 106-3 x 106 cells were removed from the cell culture, centrifuged and resuspended in cryopreservation medium (5.5% v / v Dextran-40, 10% v / v HSA, 5% v / v DMSO). The cells were frozen in a controlled speed freezer and transferred to liquid phase nitrogen storage for cryopreservation. Approximately 1ml_ cells per bottle were cryopreserved at a concentration ranging from 106 - 107 per ml_. The remaining crop was taken to the final point (day 35), referred to as "fresh (no cryopreservation in process)". Phenotypic analyzes were performed on day 21, 28 and 35 of the cell culture. The in vitro functionality (cytotoxicity K562, E: T 10: 1) was evaluated at day 35 (end point) of the culture.

The post-thawing operation of the cryopreserved culture samples in process (day 9 and 14) were cultured in the following manner. The cell bank flasks were thawed rapidly in the 37 ° C water bath. The cells were then diluted with RPMI-FBS medium, centrifuged, resuspended and seeded in culture medium. Each of the cultivation conditions was carried forward to day 35, cumulative from the beginning of the cultivation process. Analytics (cell counting, viability, phenotypic analysis, functionality evaluation) were done in the same way as their "fresh" counterparts.

Results Cryopreserved samples in process of day 9, 10 and 14 all produced excellent post-thawing viability, regardless of the point of time in process or cellular concentration (96.2% -97.3% blue triptan negative, 86.1% -93.2% Annexin-V negative / TO-PRO 3 negative). The banking in process had no negative effects on loss of final crop yield (day 35). The final cellular yield between day 9 or day 14 post-thawing and "fresh" conditions are comparable, with HS-AB or HS-AB as the source of serum.

The phenotypic profiles of NK cells maturing at day time points 21/28/35 were quite comparable between the "fresh" and "post-thaw" cultures, respectively. The phenotypic purity (CD56 + CD3) was comparable as well. The expression of certain functional NK markers (CD94, NKG2D) were slightly different due to variabilities.

In the K562 cytotoxicity assay, post-thawing NK cells grown on day 9/14 were found to produce -0-20% lower specific K562 lysis reading compared to non-processed cryopreserved NK cells. However, given the expression levels of surface markers relevant to NK cytotoxic function (DNAM1, NKp46, NKG2D, etc.) were not reduced concurrently, the trend would need to be confirmed with additional donors and test repetitions. Overall, cryopreservation in process on day 9/14 of cultivation had minimal impact on the outcome of the process. 7. 14. Example 14: Development of post-thawing medium for NK cell dosing This example demonstrates the development of post-thaw medium for NK cell dosing in animals. The effects of injection medium and cell density were tested on NK cell viability, cytotoxicity, cell recovery and lump formation. The viability was evaluated by spotting with tryptan blue; The cytotoxicity was evaluated by FACS (10: 1 ratio of NK: K562) and the formulation of the lump was evaluated by microscopic assay. The results are shown in tables 9-12 for the various types of injection medium and cell density.

Table T. Cell recovery, viability, cytotoxicity and lump formation of specific conditions tested Table 10. Cell recovery, viability, cytotoxicity and lump formation of specific conditions tested Table 11. Cell recovery, viability, cytotoxicity and lump formation of specific conditions tested Table 12. Cell recovery, viability, cytotoxicity and lump formation of specific conditions tested The results showed that the injection medium of Plasmalite + 1% HSA maintained NK cells with better viability and cytotoxicity than PBS injection medium + 1% FBS. The cytotoxicity also decreased over time after suspending the cells in injection medium. There is no effect of cell density observed in viability and cytotoxicity when the cells were suspended in Plasmalita + 1% HSA. The NK cells were also established in PBS + 1% FBS or Plasmalite + 1% HSA medium after 1 hour; however, the cells did not appear to be added. Finally, there was no obvious loss of cellular recovery and viability observed from the freeze-thaw process. 7. 15. Example 16: Development of Post-deconditioning medium for NK cell dosing The effects of various concentrations of HSA were also tested on NK cell viability, cytotoxicity, cell recovery and lump formation. The same methods were used from Example 19. The results are shown in Tables 14-16 for the various types of injection medium and cell density.

Table 13. Cell recovery, viability, cytotoxicity and lump formation of specific conditions tested Table 14. Cell recovery, viability, cytotoxicity and lump formation of specific conditions tested Table 15. Cell recovery, viability, cytotoxicity grouping of specific conditions tested Table 16. Cell recovery, viability, cytotoxicity and lump formation of specific conditions tested The results show that the viability remained well over 4 hours post-thawing in the three injection media tested. Also, the cytotoxicity decreased with time in three injection media. However, the level of reduction was lower in higher concentrations of HSA while Plasmalita + 5% HSA maintained the highest cytotoxicity. It was also observed that NK cells do not aggregate in all three injection media. Overall, Plasmalite appears to be a better candidate for injection medium than PBS. 7. 16. Example 17: Culture of NK cells without IL-2 This example demonstrates the culture of NK cells in the absence of IL-2. Cell culture was performed by the two-step process described in Example 11. Five different concentrations of IL-2 in the first medium were tested: 0, 200, 500, 1000, 2000 U / mL.

The results indicate that developing NK cells in culture did not appear to respond to growing IL-2. The purity of NK cells did not seem to depend on IL-2; NK cells differentiate into CD56 + CD3- phenotype in the absence of IL-2. The combination of IL-7, IL-15 and SCF appeared to be sufficient for cell development NK in vi tro.

Equivalents: The present invention should not be limited in scope by the specific embodiments described herein. In fact, various modifications of the invention in addition to those described will be apparent to those skilled in the art from the following description and accompanying figures. Such modifications should fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same degree as if each individual publication, patent or patent application individually and individually was indicated for be incorporated by reference in its entirety for all purposes. The citation of any publication is for description before the filing date and should not be considered an admission that the present invention should not precede said publication by virtue of the prior invention.

Claims (25)

1. - A method of producing a population of natural killer cells (NK), comprising: (a) planting a population of hematopoietic stem or progenitor cells in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of the stem cell factor (SCF) and interleukin-7 (IL-7) , wherein said optional IL-15 and SCF and IL-7 are not comprised within an undefined component of said medium, such that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of cells hematopoietic stem or progenitors differentiate into NK cells during said expansion; and (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.

2. A two-step method of producing a population of natural killer (NK) cells, wherein a first step of said method comprises expanding a population of hematopoietic stem or progenitor cells into a first medium comprising one or more of the stem cell factor (SCF), interleukin-7 (IL-7) and interleukin-15 (IL-15), and wherein said SCF, IL-7 and IL-15 are not comprised within an undefined component of said medium, and in where a plurality of hematopoietic stem or progenitor cells within said population of stem cells or Hematopoietic progenitors differentiate into NK cells during said expansion; Y wherein a second step of said method comprises expanding the cells from the first passage into a second medium comprising interleukin-2 (IL-2), to produce activated NK cells.

3. - The method according to claim 1, wherein the first medium further comprises one or more of tyrosine kinase 3 ligand type Fms (Flt3-L), thrombopoietin (Tpo), interleukin-2 (IL-2) or heparin.

4. - The method according to claim 3, wherein the first medium further comprises fetal bovine serum or human serum.

5. - The method according to claim 3, wherein the SCF is present at a concentration of about 1 to about 150 ng / mL in the first medium.

6. The method according to claim 3, wherein the Flt3-L is present at a concentration of about 1 to about 150 ng / mL in the first medium.

7. The method according to claim 3, wherein the IL-2 is present at a concentration of about 50 to about 1500 IU / mL in the first medium.

8. The method according to claim 3, wherein the IL-7 is present at a concentration of about 1 to about 150 ng / mL in the first medium.

9. The method according to claim 3, wherein the IL-15 is present at a concentration of 1 to about 150 ng / mL in the first medium.

10. The method according to claim 3, wherein the IL-15 is present at a concentration of 1 to about 150 ng / mL in the first medium.

11. - The method according to claim 3, wherein the TPO is present at a concentration of about 1 to about 150 ng / mL in the first medium.

12 -. 12 - The method according to claim 1, wherein said IL-2 in the second step is present at a concentration of 50 to about 1500 IU / mL in the second medium.

13. - The method according to claim 1, wherein said second means further comprises one or more of fetal calf serum (FCS), transferin, insulin, ethanolamine, oleic acid, linoleic acid, palmitic acid, bovine serum albumin (BSA) ) and phytohemagglutinin.

14. - The method according to claim 1, wherein the hematopoietic stem or progenitor cells are CD34 +.

15. The method according to claim 1, wherein the haematopoietic stem or progenitor cells comprise hematopoietic stem cells or progenitors of human placenta perfusate and umbilical cord hematopoietic progenitor or stem cells, wherein said placental perfusate and said umbilical cord are from the same placenta.

16. The method according to claim 1, wherein the feeder cells in step (b) comprise peripheral blood mononuclear cells treated with mitomycin C (PBMC), K562 cells or tissue culture adherent stem cells.

17. - The method according to claim 1, wherein the NK cells are CD3"CD56 + CD16".

18. - The method according to claim 17, wherein the NK cells are additionally CD94 + CD117 +.

19. - The method according to claim 17, wherein the NK cells are additionally CD161".

20. - The method according to claim 17, wherein the NK cells are additionally NKG2D +.

21. - The method according to claim 17, wherein the NK cells are additionally NKp46 +.

22. - The method according to claim 17, wherein the NK cells are additionally CD226 +.

23. - A population of activated NK cells obtained by a method comprising: (a) planting a population of stem cells or hematopoietic progenitors in a first medium comprising interleukin-15 (IL-15) and, optionally, one or more of the stem cell factor (SCF) and interleukin-7 (IL-7) , wherein said optional IL-15 and SCF and IL-7 are not comprised within an undefined component of said medium, such that the population expands, and a plurality of hematopoietic stem or progenitor cells within said population of cells hematopoietic stem or progenitors differentiate into NK cells during said expansion; Y (b) expanding the cells of step (a) in a second medium comprising interleukin-2 (IL-2), to produce a population of activated NK cells.

24. A method of suppressing the proliferation of tumor cells comprising contacting the tumor cells with a plurality of NK CD94 + CD117 cells wherein said NK cells have been produced by the method according to claim.

25. The method according to claim 24, wherein said tumor cells are primary ductal carcinoma cells, glioblastoma cells, leukemia cells, acute T cell leukemia cells, chronic myeloid lymphoma (CML) cells, leukemia cells acute myelogenous, chronic myelogenous leukemia (CML) cells, lung carcinoma cells, colon adenocarcinoma cells, histiocytic lymphoma cells, multiple myeloma cells, colorectal carcinoma cells, colorectal adenocarcinoma cells, prostate cancer cells or cells of retnoblastoma.